Acta Anatomica Sinica

Preparation and biocompatibility of acellular matrix grafts of human foreskin

LIN Jun-shan ZHAO Xin LI Du-miao XU Ya-li

2020, 51 (3): 446-449. doi: 10.16098/j.issn.0529-1356.2020.03.023

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Objective To prepare human foreskin tissue acellular matrix graft(AMGs), and explore the toxicity of the extracts on rabbit urethral mucosal cells. Methods After preparing the decellularized matrix of human foreskin tissue, the characteristics of decellularized matrix were observed by Masson staining, and the pore size was observed by scanning electron microscope.

Results Masson staining showed that the cells were removed, and the scaffold was composed of collagen. Scanning electron microscopy showed that the pore size of the scaffold was about 20-50 μm, which was uniformly distributed. The appropriate concentration of AMGs matrix extract had no obvious toxicity to urethral mucosal cells. Conclusion The acellular matrix of human foreskin tissue is an ideal AMGs scaffold material. The prepared AMGs material has low cytotoxicity and has practical value.

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Distribution, function and pathologic impacts of cardiac lymphatic vessels#br#

WANG Hai-jie TAN Yu-zhen

2020, 51 (3): 469-472. doi: 10.16098/j.issn.0529-1356.2020.03.027

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Drainage of lymph plays an important role in maintaining homeostasis of the myocardium. In heart diseases such as myocardial infarction and heart failure, injure or dysfunction of the lymphatic vessels result in cardiac lymphedema, leading to cardiac fibrosis, inflammation and cardiac dysfunction. In recent years, more attention has been put on studying relation of cardiac lymphedema with heart diseases and physiopathologic impacts of cardiac lymphangiogenesis. Targeting cardiac lymphangiogenesis is regarded as a feasible therapy for relieving cardiac lymphedema. However, the optimized strategies to sustainedly release growth factors or drugs and to transplant stem/progenitor cells need to be investigated. This article reviews mainly the characteristics of the distribution and function of the cardiac lymphatic vessels, and discusses the pathologic affects of cardiac lymphedema, the mechanisms of cardiac lymphangiogenesis and clinical impacts of promoting cardiac lymphangiogenesis.

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Coronin，a novel potential target in tumor

LIN Chen MA Qi WANG Ping

2020, 51 (3): 461-468. doi: 10.16098/j.issn.0529-1356.2020.03.026

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Coronin, a protein has a coronal-like pattern and locates below the cell membrane. Owing to its particular structure, coronin plays a key role in embryonic development and epithelial cell migration via its binding to microfilaments. Recently, proteins in coronin family were demonstrated that they could regulate the proliferation and migration of tumor cells through a series of molecular mechanisms, and there was a positive relationship between its abnormal expression and prognosis in the clinical patients. Therefore, its function and role in the progress of tumorigenesis is a hot topic in the current field of tumor research. In this paper, we made a comprehensive description of its molecular structural features, summarized its different roles in tumors, and predict its potential therapeutic prospects in the future.

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Transfection of microRNA-21 mediating the biological behavior and cellular radiosensitivity of esophageal squamous carcinoma cells

SHAO Zhi-xiong XIE Jun-jie XU Zhen-hua ZENG Cheng SONG Qing-ling YI Feng-tao

2020, 51 (3): 385-391. doi: 10.16098/j.issn.05290-1356.2020.03.012

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Objective To study the biological properties of microRNA(miRNA)-21 expression in esophageal squamous cell carcinoma.

Methods MiRNA-21 sense expression vector, miRNA-21 empty vector and miRNA-21 inhibitor were transfected and detected by fluorescence Real-time PCR. The normal cells of esophageal squamous cell carcinoma TE-1 were used as the control group, and the transfection result were detected. The cell proliferation activity was detected by cell counting kit-8(CCK-8), and the apoptosis ability was detected by flow cytometry. The invasion ability and migration ability of TE-1 cells were observed by Transwell chamber and scratch test. Changes in the sensitivity of TE-1 cells to radiotherapy were observed by colony formation experiments. The data were analyzed using SPSS 19.0 software. Results The result of Real-time PCR showed that the expression level of miRNA-21 in TE-1 cells was significantly decreased after transfected with miRNA-21 inhibitor (P<0.05). Compared with the normal cell growth group, positive control group and negative control group, the proliferation of TE-1 cells was reduced and apoptosis was accelerated after transfection with miRNA-21 inhibitor (P<0.05). In addition, the ability of cell invasion and migration in the experimental inhibition group was decreased. Clone formation assays showed that inhibition of miRNA-21 expression significantly increased the sensitivity of TE-1 cells to radiation. Conclusion Down-regulation of miRNA-21 reduces the proliferation, invasion, migration and sensitivity of radiotherapy in esophageal squamous cell carcinoma TE-1 cells, and is a potential target for the future treatment of esophageal cancer.

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Effects of microRNAs on invasion, migration and apoptosis of lung cancer cell lines

HAN Jing-jun ZHANG Yan-wei ZHANG Lei XU Ying-jie LI Hai-zhou LIU Shu-guang

2020, 51 (3): 392-397. doi: 10.16098/j.issn.0529-1356.2020.03.013

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Objective To investigate the effects of microRNAs(miRNAs) on invasion, migration and apoptosis of lung cancer cell lines.

Methods Target cells were infected with hsa-mir-933, hsa-mir-4700-3p, hsa-mir-3144-3p, hsa-mir-3972, and hsa-mir-548a-5p. Cell apoptosis was measured using flow cytomertry, Transwell invasion assay,cell migration were analyzed by lineation,miRNA was quantified using Real-time PCR. Results Compared with the vector and control, in hsa-mir-933, hsa-mir-4700-3p, hsa-mir-3144-3p, hsa-mir-397 and hsa-mir-548a-5p group the apoptosis significantly increased,cell invasion ability significantly decreased,cell migration ability significantly decreased, miRNAs expression significantly increased. Conclusion Five microRNAs can promote the apoptosis of lung cancer cell lines, change the high invasiveness of lung cancer cell lines, inhibit the migration of lung cancer cell lines, and increase the expression of corresponding miRNAs.

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Effect of PHLDA2 on proliferation, migration, invasion and apoptosis in hepatocellular carcinoma cells

MA Zhan JIANG Zhong-xiang JIANG Zheng

2020, 51 (3): 398-404. doi: 10.16098/j.issn.0529-1356.2020.03.014

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Objective To observe the expression of pleckstrin homology like domain family A member 2 (PHLDA2) in hepatocellular carcinoma (HCC) and investigate the effects of PHLDA2 on cell proliferation, migration, invasion and apoptosis. Methods To analyze the expression of PHLDA2 in 369 cases of HCC tissues and 160 cases of adjacent normal tissues and the effect of PHLDA2 expression on overall survival rate of HCC patients by gene expression profiling interactive analysis (GEPIA) online database. Cell proliferation was determined by cell counting kit-8 (CCK-8) and colony formation assay. Invasion and migration were detected by Transwell assay. The percentage of apoptosis was measured by flow cytometry. The levels of Bax and cleaved Caspase-3 were measured by Western blotting. Results The expression of PHLDA2 was upregulated in HCC, and high expression of PHLDA2 reduced the overall survival of HCC patients. Low-expression of PHLDA2 inhibited proliferation, invasion and migration, and increased apoptosis of HCC cells. Conclusion PHLDA2 promotes the occurrence and development and may act as a tumor promoter in HCC.

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Effect of heterotopic ossification on cervical motion following Prestige artificial cervical disc replacement

XU Shuai OU Yun-sheng DU Xing ZHU Yong HE Bin LI Yuan-qiang YU Hao-yang

2020, 51 (3): 405-410. doi: 10.16098/j.issn.0529-1356.2020.03.015

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Objective To analyze the effect of heterotopic ossification(HO) on the postoperative segmental range of motion（ROM）after Prestige artificial cervical disc replacement, and to explore the related factors leading to the formation of heterotopic ossification after artificial cervical disc replacement. Methods We recruited of 66 patients who had Prestige artificial cervical disc replacement from January 2014 to January 2018 in Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University were retrospectively reviewed. To evaluate cervical spine X-ray, the replacement segment ROM was measured in picture,and the occurrence of HO was defined by the McAfee’s classification. The Wilcoxon signed rank test was used to analyze the relationship between heterotopic ossification formation and replacement segment ROM,the t test was used to analyze the influence between heterotopic ossification grading and replacement segment ROM. Nine clinical factors including age, gender, body mass index(BMI), number of replacement segments, preoperative ligament ossification, the preoperative disc height ratio of the target level and its adjacent level, preoperative mobility of replacement segments，postoperative mobility of replacement segments, whether to use nonsteroidal autiinflammatory drugs(NSAIDs) during perioperative period. The correlation between these nine clinical factors and the occurrence of postoperative HO was evaluated with logistic regression analysis. Results The occurrence rate of HO was 34.8％ in last follow-up. The replacement segmental ROM of the heterotopic ossification group was significantly smaller than that of the non-ectopic ossification group, and the difference was statistically significant (P<0.05)，and the McAfee grade Ⅲ-Ⅳ group was significantly less than the McAfee grade Ⅰ-Ⅱ group, and the difference was statistically significant (P<0.05).Correlation analysis showed that preoperative and postoperative ROM of the replacement segment presented statistical correlation（P＜0.05).Furthermore analysis of ROC curve showed that heterotopic ossification was more likely to occur when the degree of ROM of the replacement segment ≤8°. Conclusion HO after Prestige artificial cervical disc replacement reduces the replacement segmental ROM，especially for the McAfee grade Ⅲ-Ⅳgroup.The occurrence of heterotopic ossification after Prestige artificial cervical disc replacement is related to the preoperative and postoperative mobility of the replacement segment. The ROM≤8° of the postoperative replacement segment is more likely to occur heterotopic ossification.

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Morphology of the fifth metacarpal bone based on three-dimensional computed tomography image and its clinical significance

DENG Jiu-zheng ZHAO Zhe ZHU Jian-Jin CHENG Xiao-guang MA Yi-min PAN Yong-wei

2020, 51 (3): 411-415. doi: 10.16098/j.issn.0529-1356.2020.03.016

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Objective To discuss the morphological features of the fifth metacarpal bone and clinical significance by measuring the three-dimensional (3-D) reconstruction model, and to improve the clinical technique and surgical instruments. Methods A total of 114 3-D reconstruction models based on computed tomography data of fifth metacarpal bone were created. The length, the radius of head, the radius of arc, the 1/2 central angle of arc, the distance of head offset, the ratio of head offset, the inner diameter of the isthmus of medullary space were measured based on 3-D models. Results The fifth metacarpal bone length was（51.55±3.01）mm. The radius of the fifth metacarpal head was (6.59±0.49）mm. The radius of the fifth metacarpal bone arc was（99.58±26.83）mm. The 1/2 central angle of fifth metacarpal bone arc was（15.90±3.36）°. The distance of the fifth metacarpal head offset was（0.49±0.26）mm. The ratio of head offset was（7.4±3.9）%. The inner diameter of the isthmus of medullary space was (2.28±0.77）mm. Conclusion The length, the radius of head, the distance of head offset, the inner diameter of the isthmus of medullary space are different between male and female, and the other parameters are not different. There is no significant difference between left and right hand.

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Anatomical and ultrasound observation of the nearby of the anterior superior iliac spine: the lateral femoral cutaneous nerve

MA Xiao-hui ZHENG Yan-yan XU Zhao-yang ZHANG Ming XU Sheng-chun

2020, 51 (3): 416-419. doi: 10.16098/j.issn.0529-1356.2020.03.017

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Objective Meralgia paresthetica(MP) is commonly caused by mechanical entrapment of the lateral femoral cutaneous nerve (LFCN). The entrapment often occurs at the site where the nerve exits the pelvis. Its optimal surgical management remains to be established, partly because the fine architecture of the fascial planes around the lateral femoral cutaneous nerve has not been elucidated. The purpose of this study is to determine the fascia structure of lateral femoral cutaneous nerve nearby the anterior superior iliac spine using anatomy and ultrasound technique. Methods Eleven cadavers were selected for plastination (6 female, 5 male, age range 38-97 years). Ultrasonography was performed on 34 healthy volunteers (19 women, 15 men, age range, 20-62 years). Results The lateral femoral cutaneous nerve exited the pelvis via a tendinous canal within the internal oblique-iliac fascia septum,and then between the sartorius muscle surface and the tensor fascia lata muscle located below the anterior superior iliac spine (ASIS). and then ran between the sartorius muscle and the tensor fascia. Conclusion Lateral femoral cutaneous nerve is located in the aponeurosis of the intra-abdominal oblique muscle at the pelvic outlet. Lateral femoral cutaneous nerve travels on the surface and outside of the sartorius muscle. These two segments of lateral femoral cutaneous nerve can be localized by ultrasound scans.

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Anatomical imaging analysis of screw configuration for femoral neck fracture

YUAN Jian-feng ZHU Qiu-liang SHI Ben-gong

2020, 51 (3): 420-424. doi: 10.16098/j.issn.0529-1356.2020.03.018

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Objective To investigate the optimal screw configuration for internal fixation of femoral neck fractures based on anatomic analysis on radiologic imaging. Methods From January to February 2017, thirty proximal femurs of 15 normal adults from Picture Archiving and Communication Systems（PACS） of Paople’s Hospibal of Anji were constructed by CT. 8 males and 7 females with a mean age of (43±8.5) years (ranging from 28 to 63 years).The medial femoral neck sections (FNS) were projected on the lateral femoral trochanteric wall. The simulated three screw configurations in the projection of FNS include: two inverted equilateral triangles symmetrized to the axis of the FNS (IET-FNS group) or the coronal axis of the proximal femur (IET-PF group) and an obtuse triangle (OT group).The distance between the screws, the distance between the centre of the FNS and the screws, and the area ratio of the triangle/FNS were calculated. Results The projection of the FNS on the lateral femoral trochanteric wall was displayed as a rotating forward ellipse. Measurements of distance between screws：OT group（5.96±0.37）cm；IET-FNS group（4.74±0.39）cm；IET-PF group（4.58±0.39）cm. Distance between the centre of the FNS to the screws：OT group（3.43±0.18）cm；IET-FNS group（2.81±0.17）cm；IET-PF group（2.72±0.14）cm. And the area ratio of triangle/FNS OT group（0.31±0.01）；IET-FNS group（0.21±0.02）；IET-PF group（0.16±0.01） were significant larger in the OT group than in the IET-FNS and IET-PF groups (P<0.05). The values of the three parameters in the IET-FNS group were also larger than those in the IET-PF group (P<0.05). Conclusion The obtuse triangle screw configuration displays advantages with respect to the parameters of distance between screws, distance between the centre of FNS to screws, and the triangle area. Therefore, the obtuse triangle screw configuration may be the ideal pattern for internal fixation of femoral neck fractures. This needs to be corroborated with biomechanics testing.

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Expression of inflammatory factors in the adipose tissues of Lepob/ob mice

SONG Ying ZHANG Han-si LI Xi-xi MA Meng-xue WANG Shuai LIN Jun-tang QIAO Liang YAN Xin

2020, 51 (3): 425-430. doi: 10.16098/j.issn.0529-1356.2020.03.019

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Objective To investigate the effect of obesity on the expression of inflammatory factors, and to explore the molecular mechanism of inflammatory factors in the adipose tissue. Methods Twenty Lepob/ob mice were randomly selected as the research object, and the wild type C57BL/6 J mice of the same nest and age as the control group. The body weight, fat mass, blood glucose level, glucose tolerance and insulin tolerance were measured. HE staining was performed to examine the morphological changes in the both white and brown adipose tissues. Western blots were conducted to analyze the protein expression of inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-κB), protein kinase B (Akt) and p-Akt. Furthermore, Real\|time PCR was performed to measure the mRNA expression of CC chemokine Iigand 2 (CCL2), CD44, colony stimulating factor 2(CSF2), glial fibrillary acidic protein (GFAP), Iba1, interleukin (IL)-1α, IL-6, IL-7, JUN and S100β in different adipose tissues. Results Comparing to the control group, the body weight of Lepob/ob mice increased significantly (P<0.001). Both fat mass and blood glucose level increased significantly (P<0.01, P<0.01, P<0.01), the tolerance of glucose was low(P<0.001) and insulin resistance was produced (P<0.001). Histological analysis of adipose tissue from Lepob/ob mice revealed an increased lipid accumulation and diameter of adipocytes compared to wild type littermates. The macrophage infiltration was also observed in adipose tissues. The expression of Janus kinase (JAK2), p-JAK2, iNOS, NF-κB, Akt and p-Akt in adipocytes of Lepob/ob mice increased significantly (P<0.05), and the expression of CCL2, CD44, IL-1α, IL-6, IL-7, JUN and S100β in the Lepob/ob mice increased significantly. Conclusion Obesity induces the significant expression of inflammatory factors in adipose tissue, which leads to disorder of cell secretion and conduction, and ultimately results in inflammatory cascade.

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Effect of alkaline phosphatase liver/bone/kidney overexpression on ventricular remodeling after acute myocardial infarction in mice#br#

WANG Li-you ZHANG Dong-ying

2020, 51 (3): 431-436. doi: 10.16098/j.issn.0529-1356.2020.03.020

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Objective To investigate the effect of alkaline phosphatase liver/bone/kidney (ALPL) overexpression on ventricular remodeling after acute myocardial infarction (MI). Methods Thirty six 8-week-old male C57BL/6 J mice were randomly divided into sham group（sham）, MI group (MI+Adv-EGFP) and ALPL overexpression group (MI+Adv-ALPL) with 12 mice in each group. Two weeks after MI，cardiac function of mice was detected by echocardiography, pathological changes was detected by HE staining. ALPL mRNA expression in mouse heart was detected by Realtime PCR. The levels of ALPL, α-smooth muscle actin (α-SMA)，collagen Ⅰ (ColⅠ) and collagen Ⅲ (ColⅢ) protein were detected by Western blotting. The collagen volume fraction (CVF) and the ratio of ColⅠ/ColⅢ were measured by polarizing method with Sirius red staining. Results Compared with MI+Adv-EGFP group, the left ventricular ejection fraction (LVEF) (%) and left ventricular fractional shortening (LVFS) (%) of the heart in MI+Adv-ALPL group were reduced significantly (P<0.05). Hearts in the ALPL overexpression group showed myocardial fiber broken, and disordered and fibrous scars were obvious in myocardial infarction area. The expression of ALPL and the expression of α-SMA, ColⅠ and ColⅢ protein in MI+Adv-ALPL group increased significantly (P<0.05), and the proportion of ColⅠ/ColⅢ in MI+Adv-ALPL group increased significantly (P<0.05).Compared with MI+Adv-EGFP group, the CVF and the proportion of the ratio of ColⅠ/ColⅢ in the MI+Adv-ALPL group increased significantly (P<0.05). Conclusion ALPL overexpression can promote the ventricular pathological remodeling after acute myocardial infarction in mice.

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Mitochondrial proteomics in myocardial hypertrophic preconditioning mice

XIN Kai-yue MA Lei-lei DONG Zhen MA Xiu-rui SUN Ai-jun

2020, 51 (3): 378-384. doi: 10.16098/j.issn.0529-1356.2020.03.011

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Objective To investigate the molecular protective mechanisms of myocardial hypertrophic preconditioning by mitochondrial quantitative proteomics. Methods Fourteen C57BL6/J male mice were randomly divided into sham group(n=6) and cardiac hypertrophy preconditioning group(n=8). The murine model of cardiac hypertrophy preconditioning was established by imposing transverse aortic constriction for 3 days and debanding the aorta for 4 days. Three mice from sham group and four mice from cardiac hypertrophy preconditioning group were randomly selected for proteomic analysis, and the remaining mice were used for functional and morphological experiments. The cardiac function was detected by echocardiography, and mechanical properties of cardiomyocytes were assessed using a SoftEdge Myocam. Cardiac morphology and mitochondrial ultrastructure were detected by pathological sections and transmission electron microscopy. The most significant mitochondrial proteins were screened by label-free quantitative proteomics and analyzed by bioinformatics analysis. Western blotting was used to verify the expression changes. Results Compared with the sham group, there were no significant changes in cardiac function and myocardial tissue morphology in the cardiac hypertrophy preconditioning group. However, electron microscopy analyses showed that the density of mitochondrial cristae increased in cardiac hypertrophy preconditioning group. Proteomic analysis screened 20 differentially expressed mitochondrial proteins. Bioinformatics analysis revealed that differentially expressed proteins were mainly related to mitochondrial ribosomal proteins. Western blotting results of key proteins were consistent with proteomic analysis. Conclusion Myocardial hypertrophic preconditioning can promote the energy metabolism of myocardial mitochondria, which may be related to the transcription, processing and transportation of mitochondrial oxidative phosphorylation complex mediated by mitochondrial ribosomal proteins.

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Effects of resveratrol pretreatment on activation and inflammation of astrocytes after oxygen-glucose deprivation/reoxygenation injury in vitro

LIU Jing LIU Jie LIAO Hong-yan CHEN Yue XIANG Qin YANG Qin

2020, 51 (3): 313-319. doi: 10.16098/j.issn.0529-1356.2020.03.001

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Objective To investigate the effects of resveratrol pretreatment on activation and inflammation of astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. Methods Twenty newborn SD rats within 24 hours were recruited,and their primary cultured astrocytes were deprived of oxygen glucose fpr 150 minutes and reoxygenation for 24 hours.The experment was divided into the normal (Nor),the control (Ctrl) and resveratrol pretreatment (Res) groups.Cell viability was detected with CCK-8 assay.Cell proliferation was measured with 5-ethynyl-2,-deoxyuridine (EdU).The expressions of glial fibrillary acidic protein (GFAP) and S100βproteins were determined with immunofluorescence.Theexpressions of GFAP,S100β,vimentin,interleukin (IL)-10,β-interferon (IFN-β),tumor necrosis factor α(TNF-α)and IL-1 β proteins were detected with Western blotting.The levels of IL-10,IFN-β,TNF-αand IL-1βproteins in the supernatant were measured by ELISA. Results CCK-8 assay showed that with the increase of resveratrol concentration (5, 20 and 40 μmol/L), the cell viability increased gradually, which was significantly lower than that of the control group (P<0.01). After that, with the increase of resveratrol concentration (80 and 100 μmol/L), the cell viability decreased significantly, and the cell viability of 100 μmol/L resveratrol group was significantly lower than in the control group (P<0.02). EdU testing showed that after OGD/R injury, the percentage of EdU positive cells in the control and resveratrol group was significantly higher than that of in the normal group (P<0.01), while it was significantly lower in the resveratrol pretreatment group than that of the control group (P<0.01).Immunofluorescence, Western blotting and ELISA method showed that the expressions or levels of GFAP, S100β, vimentin, IL-10, IFN-β, TNF-α, IL-1β proteins in the control and resveratrol group were significantly higher than that of the normal group (P<0.05), but the expresions or levels of IL-10 and IFN-β proteins in the resveratrol group were higher than those of the control group, and the others were lower than the control group (P<0.05).However，the expressions or levels of IL-10 and IFN-β proteins in the resveratrol group were higher than those of the control group, and the others in the resveratrol group were lower than those of the control group (P<0.05). Conclusion Resveratrol pretreatment can inhibit the activation of astrocytes and reduce the inflammation after OGD/R injury in vitro.

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Effects of resveratrol on microglia polarization after oxygen glucose deprivation/reoxygenation injury in vitro

LIU Jie REN Yu CHEN Yue LIU Jing XIANG Qin YANG Qin

2020, 51 (3): 320-325. doi: 10.16098/j.issn.0529-1356.2020.03.002

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Objective To investigate the effect of resveratrol on microglia polarization after oxygen glucose deprivation/reoxygenation (OGD/R) in vitro. Methods N9 microglias were cultured under oxygen-glucose deprivation for 150 minutes and reoxygenation for 24 hours.The experiment was divided into normal, control and resveratrol pretreatment groups. Cell viability was measured by cell counting kit-8(CCK-8) assay. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity in the supernatant were detected by thiobarbituric acid(TBA) and water soluble terazolium salt(WST-1) respectively. Nuclear factor E2-related factor 2 (Nrf2) nuclear translocation was detected by immunofluorescence. The levels of CD206, inducible nitric oxide synthase (iNOS), Nrf2, heme oxygenase-1(HO-1), NAD(P)H: quinone oxidoreductase 1(NQO1) protein were measured by Western blotting. The mRNA levels of CD206, iNOS were detected by Real-time PCR. Results After OGD/R injury, cell viability, SOD activity, CD206, Nrf2, HO-1, NQO1 protein or mRNA expression in resveratrol groups were significantly higher than those in the control group (P<0.05, n=3), while MDA content and iNOS protein and mRNA expression were significantly lower than those in the control group (P<0.05, n=3). Nrf2 protein in resveratrol group was significantly transferred to the nucleus compared with the control group. Conclusion Resveratrol pretreatment may regulate M1/M2 polarization of microglia and reduce oxidative stress after OGD/R via strengthening activation of Nrf2.

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Relationship between depression-like behavior and expression of aquaporin 4 in brain of urea transporter B null mice

YUAN De-zhi HOU Liang-juan TIAN Kuan HU Ling LI Jin-fang RAN Jian-hua

2020, 51 (3): 326-331. doi: 10.16098/j.issn.0529-1356.2020.03.003

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Objective To investigate the ultrastructural changes of hippocampus in urea transporter B (UT-B) null mice and the alterations of distribution and expression level of aquaporin 4（AQP4）in brain, and to discuss the relationship between AQP4 expression changes and depression-like behaviors in UT-B null mice. Methods Behavior differences of wild-type and UT-B null mice（10 in each group） were detected with sucrose preference and forced swimming test. The ultrastructural changes of hippocampus were observed by transmission electron microscopy (TEM). Immunohistochemistry and Western blotting were performed to detect the distribution and expression level of AQP4 in both genotypes. Results The sucrose preference index of wild-type mice and UT-B null mice were (84.67±1.62)% and (65.67±2.66)%, respectively (P<0.001). The immobility time of forced swimming was (209.1±7.00) seconds and (128.6±3.75) seconds respectively (P<0.001). The two behavioral test results showed that UT-B null mice exhibited depression-like behavior. TEM results displayed the abnormal neurons with swelling of myelinated and unmyelinated fibers and degenerative changes, and perivascular astrocyte endfeet swelling. Immunohistochemistry results showed AQP4-immunoreactive (IR) cells were significantly reduced in cortex, hippocampus and thalamus. AQP4-IR cells were distributed in the pia matter, ependymal and cerebrovascular, but the perivascular immunostaining decreased. Western blotting analysis showed that the expression level of AQP4 in hippocampus was down-regulated by 27.1% (P<0.05). Conclusion Reduced expression of AQP4 in the cerebral cortex and hippocampus of UT-B null mice might induce depressive behaviors by inference neurogenesis and cerebral metabolism.

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Dynamic expression of insulin-like growth factor 2 during critical period of visual development in lateral geniculate body of mice

LUO Shi-shi SU Qiang SUN Qiu-min XU Jing CAO Wen-yu XU Yang LIU Zheng-hai WAN Wei

2020, 51 (3): 338-343. doi: 10.16098/j.issn.0529-1356.2020.03.005

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Objective To detect the dynamic expression of insulin-like growth factor 2 (IGF2) in lateral geniculate body(LGB) during the critical period of visual development. Methods Three groups of Kunming mice of different ages were selected for testing, which were 3 weeks old, 5 weeks old and 7 weeks old, twelve in each group. The forepaw-reaching reflex test was used to detect whether the visual function of the mice was normal in each group. Immunohistochemical technique was used to detect the expression of IGF2 protein and its receptor in the lateral geniculate body of normal mice at week 3, 5 and 7 postnatal, and to analyze the expression of the protein of IGF2 and its receptor in each part of the lateral geniculate body. Results The expression of IGF2 protein in the dorsal lateral geniculate nucleus decreased significantly at week 5 postnatal and increased significantly at week 7 postnatal, and increased gradually over time at week 5 and week 7 postnatal in the ventral geniculate nucleus. The expression of IGF2 receptor protein in the dorsal lateral geniculate nucleus and ventral nucleus increased significantly at week 5 postnatal, and at week 7 postnatal, the expression of IGF2 receptor decreased to week 3 level in lateral geniculate body of mice. Conclusion The expression of IGF2 and its receptor in lateral geniculate body of mice during critical period of visual development changed dynamically, and the expression patterns of IGF2 and its receptor in different parts of LGB were not completely consistent. The expression of IGF2 and its receptors may be related to the plasticity of visual development in mice.

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Permeability to bloodbrain burrier of 125Ⅰ-nerve growth factor after focal cerebral ischemia#br#

ZHENG Qian MA Xin-yu CAO Zhong-wei

2020, 51 (3): 344-351. doi: 10.16098/j.issn.0529-1356.2020.03.006

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Objective To investigate the optimal time and mechanism of nerve growth factor（NGF）crossing the bloodbrain barrier (BBB) under cerebral ischemia in rats through focal cerebral ischemia experiments in rats, and to provide a new way for the clinical application of NGF. Methods A total of 65 healthy Sprague-Dawlay (SD) male rats were prepared by using a modified suture method to prepare focal cerebral ischemia models. Rats were injected with 125Ⅰ-NGF, and the γ count was measured with a γ-ray counter to observe the changes in BBB permeability at different times of ischemia. At the time point at which BBB permeability was greatest after cerebral ischemia (3 days ischemia), the rats were used as the experimental group (n=15). Coronal sections of the rats were subjected to autoradiography, and the imaging site, area and brightness were observed. The changes of blood-brain barrier at different times of cerebral ischemia with transmission electron microscope were observed. Results With the prolongation of cerebral ischemia time, the permeability of BBB increased gradually, and the permeability of BBB was the largest after 3 days of cerebral ischemia, and then weakened gradually. With the same permeability of BBB, the γ count showed that the γ counts of the control group and the experimental group increased with the prolonged medication time. The γ counts of the experimental group at each time point were higher than the control group. Significant difference (P<0.05) was found, and the highest γ count at 4 hours. The result of autoradiography showed that the entire brain was not developed in the control group for 1 hour, and the ventricles and periventricular brain tissues were developed in 4 hours and 7 hours. The area occupied about 4.1% of the cerebral coronal section, and the brightness was obvious. The area was about 6% of the coronal section of the brain, and the brightness was strong. In addition to the development of the ventricles and periventricular brain tissues at 4 hours and 7 hours, in the right frontal lobe (FL), parietal lobe (AL) and temporal lobe, there was a large area in the medulla and the medulla, which covered 36.2% to 47.3% of the coronal section of the brain. The brightness was very strong. Conclusion BBB permeability increases with prolonged cerebral ischemia in rats, of which the permeability is the strongest at 3 days of ischemia; rats with focal cerebral ischemia at 3 days for 4 hours, 125Ⅰ-NGF enters the BBB in the largest amount, in the frontal and parietal. The temporal lobe is most obvious. NGF can pass the blood-brain barrier of focal cerebral ischemia and may become a new breakthrough in clinical drug delivery.

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Mathematical model construction and analysis of the effect of estradiol on the electrical activity of gastric antrum in female rabbits

ZHAO Jian-shuai ZHANG Liu-ning ZHANG Ying-li ZHANG Jin DU Yi-nan GUO Ya-ru LI Xiang WU Yong-jie XU Yong-ping

2020, 51 (3): 352-360. doi: 10.16098/j.issn.0529-1356.2020.03.007

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Objective To investigate the expression of estrogen receptor in gastric antrum and the effect of estrogen on electrical activity of gastric antrum smooth muscle in female rabbits in virtue of the constructed mathematical model in order to explore the regulation of estrogen on gastric motility. Methods Using immunofluorescence to observe the expression of estrogen receptor in gastric antrum. Using BL-420F bio monitor to monitor: Comparing the difference of antral electromyography index between rabbits in ovariectomized group and rabbits in sham operating group; Observing the dose-response relationship between doses of estradiol (0,0.1,0.15,0.2,0.25 and 0.3 mg/kg). To construct the mathematical model, and to analyze the dose-response relationship and mechanism of action. Rresults The expression of estrogen receptor in the antral wall of the stomach was negative. The activity index of gastric antrum myoelectric activity was significantly decreased after ovarian ablation (P<0.01). With the increase of estradiol dose, the activity index of gastric antrum muscle increased and then decreased. Analysis of variance showed that the difference of antral electromyographic activity index between adjacent groups was significant (P<0.05) or extremely significant (P<0.01). Taking the estradiol dose as the indepe ndent variable x, the antral electromyography activity index was the dependent variable y, and the fitting wass obtained: y=2.80 + 5.65 × exp{ -0.5 × ［(x-0.159)/0.038 ］2}. It was a Gaussian function (R=0.9884, P>0.05), which indicated that the regulation of estradiol on gastric antral smooth muscle was based on the intrinsic regulation mechanism of gastric antrum itself, and had a great influence on its internal regulation mechanism. Conclusion The above results indicate that there is no estrogen receptor on the gastric antrum. and the mathematical model of the dose-effect relationship of estradiol on the electrical activity index of gastric antrum in our study accords with the characteristics of Gaussian function model, which indicates that estradiol promotes the activity of gastric antral smooth muscle and is based on the stomach. It shows indirect realization of the internal regulation mechanism of sinus myoelectric activity.

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Effects of calycosin on mitochondrial apoptotic pathway in oxygen-glucose deprivation/reoxygenation PC12 cells#br#

ZHANG Xue-ning JIN Xiao-fei ZHOU Xiao-hong GAO Wei-juan

2020, 51 (3): 332-337. doi: 10.16098/j.issn.0529-1356.2020.03.004

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Objective To investigate the effect of calycosin on mitochondrial apoptotic pathway in oxygen-glucose deprivation/reoxygenation PC12 cells. Methods PC12 cells were randomly divided into four groups: control group, model group, calycosin group and nimodipine group. Except for the control group, the other groups were treated with oxygen and glucose deprivation for 2 hours and compound oxygen and glucose for 24 hours. Calycosin group and nimodipine group were treated with drug-containing medium containing calycosin (0.07 μmol/L) and nimodipine (5.00 μmol/L) simultaneously with reoxygenation. CCK-8 method was used to detect cell survival rate, flow cytometry was used to detect cell apoptosis rate, immunofluorescence method was used to detect Bax/Bcl-2 ratio, Western blotting was used to detect the expression of key proteins cytochrome C(Cyt-C), apoptotic protease activating factor-1(Apaf-1) and Caspase-3 in mitochondrial apoptotic pathway. Results Compared with the control group, the survival rate of cells in model group decreased significantly (P<0.05), and the apoptotic rate increased significantly (P<0.05), the ratio of Bax/Bcl-2 was significantly increased (P<0.05), and the expression of key proteins Cyt-C, Apaf-1 and Caspase-3 in mitochondrial apoptotic pathway were significantly increased (P<0.05). Compared with the model group, the cell survival rates of calycosin group and nimodipine group increased significantly (P<0.05), apoptotic rate decreased significantly (P<0.05), the ratio of Bax/Bcl-2 was significantly decreased (P<0.05), and the expression of key proteins of mitochondrial apoptotic pathway, Cyt-C, Apaf-1 and Caspase-3 were significantly decreased (P<0.05). The difference has statistical significance. Conclusion Calycosin can significantly improve the survival rate of oxygen-glucose deprivation/reoxygenation PC12 cells and inhibit cell apoptosis. Its mechanism is closely related to the inhibition of the expression of key proteins Cyt-C, Apaf-1 and Caspase-3 in mitochondrial apoptotic pathway by calycosin.

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Observation and analysis of 20 facial morphological characteristics of Xibo ethnicity adults in Chabchar in Xinjiang

HOU Yan-li XU Guo-chang ZHANG Qing-yuan LIU Rong-zhi XU Ming-zhu WEN You-feng

2020, 51 (3): 437-441. doi: 10.16098/j.issn.0529-1356.2020.03.021

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Objective To investigate the 20 facial morphological characteristics of Xibo ethnicity adults in Chabchar in Xinjiang, and to explore the causes of the features, so as to provide basic data for the basic study of minority anthropology. Methods A random sampling method was used to determine 677 Xibo ethnicity adults, and 20 facial morphologyical characteristics was measured and statistically analyzed. Results In terms of age groups, a total of 3 indicators which were opening height of eyeslits(χ2=27.47, P<0.05), hair shape(χ2=16.10, P<0.05) and brow ridges(χ2=13.92, P<0.05)were statistically significant in different age groups among the Xibo males; and a total of 6 indicators which were cleft gap (χ2=16.56, P<0.05), eyebrow(χ2=22.62, P<0.05), maximum diameter of nostrils(χ2=12.74, P<0.05), lower jaw(χ2=12.64，P<0.05), brow ridges (χ2=22.00，P<0.05), nasal profile(χ2=50.49, P<0.05) were statistically significant in different age groups among the Xibo females, other indicators had no statistical significance. From the perspective of gender, a total of 8 indicators which were statistically significant between males and females among the 20 features of the Xibo ethnicity adults, they were hair vortex (χ2=11.68, P<0.05), frontal slope (χ2=12.99, P<0.05), direction of eyeslits(χ2=27.67, P<0.05), ear tip(χ2=19.11, P<0.05), hair shape(χ2=87.26, P<0.05), eyebrow(χ2=30.82, P<0.05), brow ridges(χ2=33.74, P<0.05), a d Opening height of eyeslits(χ2=8.43，P<0.05), while other 12 indicators have no statistical significance. Conclusion The facial morphological characteristics of the Xibo ethnicity adults in chabchar in Xinjiang showed the unique characteristics of anthropology in the region, and the reasons for those may be related to various factors such as genetic, living environment and habits.

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Comparative analysis of serum biochemical indexes of Uygur and Kazak adults of different ages and genders in Xinjiang

YIN Jin-peng YANG Xian-guo XU Guo-chang ZHAO Xu-lin LIU Rong-zhi KULINAR Toja-da

2020, 51 (3): 442-445. doi: 10.16098/j.issn.0529-1356.2020.03.022

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Objective To analyze the changes of serum biochemical indexes with age and gender in Xinjiang Uygur and Kazak populations, and to compare the differences of serum biochemical indexes between Xinjiang Uygur and Kazak populations in gender and age. Methods Totally 511 Xinjiang Uygur and Kazak healthy adults were randomly selected from a hospital in Xinjiang Uygur autonomous region for normal physical examination, including 252 and 259 Xinjiang Uygur and Kazak healthy adults. Serum biochemical indexes were detected by CX-7 automatic biochemical analyzer produced by Beckman, USA, and statistical analysis was conducted by variance analysis, t test, correlation analysis and chi-square test. Results Xinjiang Uygur nationality and Kazak serum biochemical indexes for the male and the female, including glutamic-pyruvic transaminase(GPT), direct bilirubin (DBil), uric acid(UA), creatinine(Cre) and urea(Ure), glucose (Glu), total bilirubin（TBil）, cystatin C(Cys-C) increased with the growth of the age, and sex between different age groups were statistically significant (P<0.05), the men than women with age (P<0.05); High-density lipoprotein (HDL) decreased gradually with age, and low-density lipoprotein (LDL), triglyceride and total cholesterol increased gradually with age, with statistical differences among different age groups (P<0.05). Triglycerides(TG), total cholesterol (TC) and LDL were lower in females than in males in all age groups before the age of 50, and HDL was higher in females than in males (P<0.05), and there was no difference after the age of 50 (P>0.05). There were statistically significant differences in Glu, TG, TC, LDL and HDL between Xinjiang Uygur and Kazak people of the same ender and age group (P<0.05). Conclusion The serum biochemical indexes of Uygur and Kazak in Xinjiang are different in different ages and genders. The Glu and lipid levels of Uygur nationality in Xinjiang are different from those of Kazak nationality.

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Effect of genipin on cardiomyocyte apoptosis and autophagy in rat after hypoxia/reoxygenation injury#br#

LUO Xue-xiu LIU Jian

2020, 51 (3): 361-366. doi: 10.16098/j.issn.0529-1356.2020.03.008

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Objective To investigate the effect of genipin on cardiomyocyte apoptosis and autophagy in rat after hypoxia/reoxygenation (H/R) injury. Methods The method with hypoxia treatment of H9c2 cells for 12 hours and then reoxygenation treatment for 4 hours was used in the present study in order to establish H/R model. The H9c2 cells were divided into control group (Con), genipin group (GE), model group (H/R), model + genipin group (H/R+ GE). Cell viability was detected by cell counting kit-8(CCK-8). Cell apoptosis was determined by flow cytometry. Transmission electron microscopy was used to observe autophagosomes. The expression of Bax, Bcl-2, P62, Beclin1, LC3-Ⅱ, protein kinase B(Akt), p-Akt, mammalian target of rapamycin(mTOR), p-mTOR proteins were assessed by Western blotting. Results Genipin pretreatment enhanced the cell viability, prevented cell apoptosis and autophagosome accumulation, and reduced the ratio of the cross-sectional areas of the autophagic structures to that of the cytoplasm after H/R injury in H9c2 cells. Western blotting showed that genipin pretreatment decreased the expression of Bax, LC3-Ⅱ, Beclin1 proteins and increased the expression of Bcl-2, p62, p-Akt, p-mTOR proteins after H/R injury. Conclusion Genipin can inhibit H/R injury-induced apoptosis and autophagy, which may be through activating Akt/mTOR signaling pathway.

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Effects of dihydrotestosterone on the expression of anti-Müllerian hormone in primary follicular granulosa cells in rats

WANG Mei-ling DONG Yu-ting YE Xiao-feng HEI Chang-chun CAI Yu-fang KONG Bin ZHAO Cheng-jun CHANG Qing

2020, 51 (3): 367-372. doi: 10.16098/j.issn.0529-1356.2020.03.009

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Objective To explore how dihydrotestosterone (DHT) affects the expression of anti-Müllerian hormone (AMH) in primary rat follicular granulosa cells, and then provides a new experimental basis for the role of DHT and AMH in follicular development. Methods Granulosa cells was extracted from the ovaries of 95 21 days SD female rats for primary culture. Firstly, cell morphology were detected by HE staining and follicke stimulating hormone receptor(FSHR) immunofluorescence detected cell purity after 48 hours of cell culture. Then, cells were randomly divided into control group (without drug intervention), 10-8mol/L DHT group, 10-5mol/L DHT group, 2×10^-5 protein kinase B(Akt) inhibitor (MK-2206 2Hcl) group and 10-8mol/L DHT+ 2×10^-5 mol/L MK-2206 2Hcl group according to different intervention method and experiments，corresponding concentrations were used in each group to intervene cells for 3 hours. After intervention, sites and expression changes of AMH were detected by immunofluorescence. Changes of AMH, Akt and p-Akt were detected by Western blotting. Results Immunofluorescence showed that the purity of primary follicular granule cells in rats was 93.33%±3.09%,and AMH was expressed in the membrane and cytoplasm of primary follicular granule cells in rats; Compared with the control group, both 10-8 mol/L DHT and 10-5 mol/L DHT increased the expression of AMH in granulosa cells (P<0.05),but there was no difference in the effect of these two concentrations of DHT on the expression of AMH in granulosa cells (P>0.05); Compared with the control group, 10^-8 mol/L DHT increased p-Akt and AMH expression, and 2×10^-5MK-2206 2Hcl decreased AMH expression (P<0.05); Compared with the 2×10^-5 MK-2206 2Hcl, AMH expression was increased in the 10^-8 mol/L DHT+2×10^-5 MK-2206 2Hcl treatment group (P<0.05). Conclusion DHT can up-regulate the expression of AMH in primary follicular granulosa cells in rats, and this effect may be related to the Akt signaling pathway.

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Effect of tripterygium wilfordii polyglycoside on renal cell autophagy in rats with diabetic nephropathy and its related mechanism

ZHANG Jun DING Ting TANG Dong-xing WANG Jian-ping

2020, 51 (3): 373-377. doi: 10.16098/j.issn.0529-1356.2020.03.010

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Objective To investigate the effects of tripterygium glycosides on autophagy of renal cells in rats with diabetic nephropathy and to analyze its molecular mechanism. Methods A rat model of diabetic nephropathy was made by intraperitoneal injection of streptavidin. Thirty SD rats were randomly divided into 5 groups, control group, model group, 0.1 mg/kg drug group, 0.5 mg/kg drug group and 1.0 mg/kg drug group, and each group has 6 rat. After the successful establishment of the diabetic nephropathy model, the 0.1 mg/kg drug group, the 0.5 mg/kg drug group, and the 1.0 mg/kg drug group were intragastric administration with 0.1, 0.5, and 1.0 mg/kg tripterygium glycosides, respectively, and the control group and the model group were intraperitoneally injected with the same amount of normal saline. The levels of renal function and oxidative stress were compared among groups. The expression levels of p-mammalian target of rapamycin (p-mTOR),mammalian target of rapamycin(mTOR), microtubules associated protein 1 light chain 3β-Ⅱ/microtubules associated protein 1 light chain 3β-Ⅰ(LC3-Ⅱ/LC3-Ⅰ) and Beclin1 protein were detected by Western blotting. The expression levels of LC3, LC3-Ⅱ and Beclin1 mRNA in each group were detected by Real-time PCR. Results Compared with the control group, the serum creatinine(Scr), blood urea nitrogen(BUN), protein(Pro) and malondialdehyde(MDA) levels in the model group increased,and the the glutathione peroxidase(GSH-Px) and catalase(CAT) levels decreased significantly (P<0.05). Compared with the model group, the Scr, BUN, Pro and MDA levels of the drug group were significantly decreased, and GSH-Px and CAT levels were significantly increased in a dose-dependent manner (P<0.05). Compared with the control group, the expression level of p-mTOR protein in the renal tissue of the model group was increased, and the expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin1 protein were decreased (P<0.05). Compared with the model group, the expression level of p-mTOR protein was decreased in the dose group, and the expression levels of LC3-Ⅱ/LC3-Ⅰ protein in the dose group were significantly increased in a dose-dependent manner (P<0.05). The expression levels of Beclin1 protein in the 0.5 mg/kg drug group and 1.0 mg/kg drug group, were significantly higher than the model group (P<0.05). Compared with the control group, the expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin1 mRNA in the renal tissue of the model group were significantly lower (P<0.05). Compared with the model group, the expression levels of LC3 and Beclin1 mRNA in the drug groups of each dose group were significantly increased (P<0.05). Conclusion Tripterygium wilfordii glycosides can protect kidney function in rats with diabetic nephropathy, and its mechanism might be related to inhibition of oxidative stress and activation of autophagy.

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A model of partial ligation of common carotid artery and neointima

JIANG Zhi-long JIANG Yue SUN Wen-qing CHEN Xiao-kang ZHANG You-zhi

2020, 51 (3): 456-460. doi: 10.16098/j.issn.0529-1356.2020.03.025

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Objective To discuss a method to induce vascular remodeling (intimal regeneration) after partial ligation of the left common carotid artery. Methods Forty 8-week-old male C57BL/6 J mice were randomly divided into 6 groups: normal group (WT), sham group (sham), 4 weeks, 8 weeks, 10 weeks and 12 weeks postoperative groups. Only the internal carotid, external carotid and occipital arteries in the four distal branches of the left common carotid artery (LCA) were ligated, and the superior thyroid artery was retained, resultsing in intimal neovascularization and vascular remodeling of the left common carotid artery. The changes of body weight and forage were observed after operation. Morphological changes of blood vessels were observed by HE staining. The aggregation of collagen fibers in blood vessels was observed by sirius red staining. Results In addition to the WT group, the weight and dietary quantity of other groups were reduced 1-2 days after the operation, and began to rise from the third day, while the sham group began to rise from the second day. HE staining showed that no new intima was formed in the left common carotid artery of sham group and WT group, and thickening of intima and media layer and stenosis of the nasal cavity were observed in the 4, 8, 10 and 12 weeks groups after partial ligation. There was no intima thickening in the unligated carotid artery on the right side, but there was an enlargement of the lumen. This sirius red stain demonstrated collagen accumulation in the intima and tunica media.

Conclution Partial ligation of the left common carotid artery can establish a mouse model of arterial endothelial injury with obvious intima regeneration.

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Sudan black B reducing the intestinal autofluorescence of rats and its application in immunofluorescence

LI Yong ZHOU Li ZHANG Yang LIU Zhen YAO Nian SHEN Feng-ge WANG Zhi-yong

2020, 51 (3): 450-455. doi: 10.16098/j.issn.0529-1356.2020.03.024

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Objective To develop a feasible method to reduce the rat intestinal autofluorescence (AF). Methods Five rats were sacrificed. After cryostat section, intestinal AF was investigated by immunofluorescence (IF) before and after Sudan black B (SBB) treatment. Results The AF within the different rat intestinal segments was easily detected in both FITC- and TRITC-channel spectrum, which was not affected by serum blocking. After 0.3% SBB treatment for 10 minutes, a significant reduction of AF was observed in the rat intestinal tissue. Moreover, the SBB treatment did not affect the expression of CD45 in the rat intestine detected by IF staining, but reduced the AF remarkedly. Conclusion The AF in rat intestinal frozen section can be effectively reduced with 0.3% SBB treatment for 10 minutes.

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