Acta Anatomica Sinica

Establishment of calcium-activated chloride channel -based second messenger Ca²⁺detection method

XIAO Yun-ping XIE Yu-hao ZHANG Jia-qi GUO Jia-qi DING Xu HAO Feng WANG Guo-qing

2021, 52 (2): 311-316. doi: 10.16098/j.issn.0529-1356.2021.02.024

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Objective To establish a cell model based on calcium-activated chloride channel（CaCC）that could sensitively detect the second messenger Ca²⁺ in the cytoplasm. Methods The eukaryotic expression vectors of anoctamin 1(ANO1) and YF-H148Q/I152 L were constructed respectively. FRT cells co-expressing ANO1 and YFP-H148Q/I152 L were obtained by liposome transfection. The expression of ANO1 and YFP-H148Q/I152 L in FRT cells was observed by an inverted fluorescence microscope, and flow cytometry was used to detect the purity of cells. Patch clamp was applied to study physiological characteristics of CaCC. The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CaCC modulators was verified by the fluorescence quenching kinetics experiments. The fluorescent probe was used to detect the calcium concentration in cytoplasm after adding CaCC activator. Results The result of the inverted fluorescence microscope showed that ANO1 was expressed in the cell membrane of FRT, and YFP-H148Q/I152 L was expressed in the cytoplasm of FRT cells. The cell model had the physiological characteristics of classical calcium-activated chloride channels. The FRT cell model stably co-expressing ANO1 and YFP-H148Q/I152 L was successfully constructed. The model could screen CaCC modulators, and the slope of fluorescence change and the concentration of CaCC modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the calcium concentration in the cytoplasm. The cell model can sensitively detect intracellular calcium concentration. Conclusion The cell can efficiently and sensitively detect the second messenger Ca²⁺ concentration in the cytoplasm, and it provides a simple and efficient method for the study of other targets associated Ca²⁺ signal.

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Molecular mechanisms of palate development

XU Chen HU Xu-feng

2021, 52 (2): 317-322. doi: 10.16098/j.issn.0529-1356.2021.02.025

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The mammalian palate develops from the primary palate and the secondary palate. Development of the mammalian secondary palate involves highly dynamic morphogenetic processes, including the outgrowth of palatal shelves from the oral side of the embryonic maxillary prominences, the elevation of the initially vertically oriented palatal shelves to the horizontal position above the embryonic tongue, and the subsequent adhesion and fusion of the paired palatal shelves at the midline to separate the oral cavity from the nasal cavity. In addition to identifying a large number of genes required for palate development, recent studies have begun to unravel the extensive cross-regulation of multiple signaling pathways, including sonic hedgehog(Shh), bone morphogenetic protein(BMP), fibroblast growth factor(FGF), transforming growth factor β(TGF-β), and Wnt signaling, during palatal shelf growth and patterning. Here we summarize major recent advances and integrate the genes and molecular pathways with the morhogenetic processes of palate development.

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Advances in the application of microfluidic technology in assisted reproduction

WANG Zhong-li WANG Qi DENG Juan LEI Lan-jie YANG Lei

2021, 52 (2): 323-328. doi: 0.16098/j.issn.0529-1356.2021.02.026

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The field of assisted reproductive technology (ART) has made rapid progress. Due to the complicated procedures in clinical practice, such as sperm sorting, oocyte selection, fertilization, embryo culture and embryo monitoring, there is still a need for further improvement in efficiency and clinical decision-making. Microfluidic technology is not only a technical means but also a science, which studies the behavior characteristics of fluid at the sub-microlevel. The pipeline and detection terminal of the detection system have great flexibility and can be applied to different detection purpose. It has high detection accuracy and saves the size of samples. It has the characteristics of high integration and matches the detection needs in the practice of assisted reproduction. The application of microfluidic technology can evaluate the basic biological information such as the structure, function and environment of spermatozoa, oocytes and preimplantation embryos, and make accurate and effective coping lans and clinical decisions. This paper reviews the application of microfluidic technology in assisted reproduction, in order to promote the application and improvement of microfluidic technology in the field of reproduction.

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Astragaloside Ⅳ alleviating chronic intermittent hypoxia-induced cardiac injury by enhancing autophagy

QIN Lu-yun LUO Li-fei GUAN Peng SUN Zhi-min WANG Na

2021, 52 (2): 270-276. doi: 10.16098/j.issn.0529-1356.2021.02.017

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Objective To investigate the protective effects of astragaloside Ⅳ（ASⅣ）on chronic intermittent hypoxia (CIH)-induced cardiac injury. Methods Twenty-four male adult Sprague Dawley rats were randomly assigned to control, CIH, CIH+ASⅣ, and ASⅣ group, 6 rats in each group. Circular nitrogen and oxygen were filled to make oxygen concentration change between 9%-21% for the CIH treated rats. The exposure cycle was repeated every 3 minutes, 8 hours/day for 35 days. ASⅣ was given by intragastric administration daily before intermittent hypoxia exposure in the CIH+ASⅣ group and ASⅣ group. The control group and CIH group were given normal saline of the same quantity. Echocardiography was used to analyse cardiac function. Myocardial structure was assessed by HE and wheat germ agglutinin staining. The apoptosis of cardiomyocytes was detected by TUNEL assay. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in heart were detected by commercial kits. Western blotting was used to evaluate the levels of Bcl-2, Bax, LC3, Beclin1, P62, and mammalian target of rapamycin（mTOR）. Results In the CIH group, the left ventricular ejection fraction (LVEF) and left ventricular internal diameter at end-systole (LVIDs) were inhibited, the myocyte cells showed disordered arranged, enlarged diameters and higher apoptosis rate. The MDA content was significantly elevated and the SOD activity decreased in CIH group when compared with those of control. What’s more, the expression level of Beclin 1 decreased while the P62 expression and the p-mTOR/mTOR ratio increased in the CIH group. Compared with the model group, the LVEF, LVIDs, SOD activity, LC3Ⅱ/Ⅰ ratio, and Beclin1 expression of rats in the CIH+ASⅣ group increased. The cardiomyocytes in the rats of CIH+ASⅣ group showed normal arrangement and diameters. The apoptosis rate, MDA content, P62 expression and the p-mTOR/mTOR ratio decreased in the CIH+ASⅣ group when compared with the CIH group. Conclusion ASⅣ can alleviate CIH-induced cardiac injury by promoting autophagy via mTOR.

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Xinmailong injection improving myocardial ischemia reperfusion injury in isolated-heart of rats

WANG Yong-jie JIANG Yong-liang LIN Zhi LIU Ran JIANG Xue-mei LU Di SUN Lin

2021, 52 (2): 277-283. doi: 10.16098/j.issn.0529-1356.2021.02.018

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Objective To observe the protective effect of Xinmailong injection (XML) on myocardial ischemia reperfusion (I/R) injury in rats model. Methods The levels of SPF 60 successful acute myocardial ischemia rat models were randomly divided into control group,I/R group and XML group(Different drug concentrations 50 mg/L,100 mg/L, 200 mg/L, 400 mg/L). The control group was continuously given K-H nutrient solution for 2 hours, the other group was given respective different drug concentrations or K-H nutrient solution by Langendorff isolated heart perfusion method after ischemia 30 minutes.The following indicators were detected: Hemodynamics included heart rate (HR), left ventricular development pressure (LVDP) and ±dp/dtmax; the myocardial enzymes included creatine kinase (CK), creatine minase-MB (CK-MB), troponin-Ⅰ (Tn-Ⅰ) and myoglobin (MYO), coronary flow and myocardial infarct size were observed. The expression of Bax and Bcl-2 in each group was detected by Western blotting and immunofluorescence. Results Xinmailong injection could alleviate LVDP and ±dp/dtmax; decrease the release of CK, CK-MB, Tn-Ⅰ, MYO and the shrink area of myocardial infarction; increase the coronary flow; enhance heart rate fast and certain inhibition of myocardial apoptosis. Conclusion Xinmailong injection can alleviate the damage of cardiac function caused by myocardial ischemia/reperfusion injury.

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Outcomes of frozen-thawed embryo transfer cycles of progestin-primed ovarian stimulation protocol compared with other three ovulation hyperstimulation protocols

LI Jian-hua JIAO Ting-ting WANG Jian-ye LI Chang-shan DONG Pan CHEN Xi ZHANG Shui-wen

2021, 52 (2): 284-288. doi: 10.16098/j.issn.0529-1356.2021.02.019

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Objective To analyze the clinical outcomes of progestin primed ovarian stimulation (PPOS) compared with the other three different controlled ovarian hyperstimulation (COH) protocols in fresh embryo transfer (ET) and frozen-thawed embryo transfer (FET) cycles. Methods A total of 430 oocyte pickup cycles and 272 FET cycles were retrospectively analyzed. Number of oocytes retrieved, laboratory indexes and pregnancy outcome of FET were compared. Results The mean oocytes retrieved (11.1±7.3), fertilization rate (85.6%), cleavage rate (95.1%) and excellent embryo rate (20.2%) as well as transplantable embryo rate (4.5±3.1) of the PPOS group did not show significant differences compared with the other 3 subgroups (all P<0.05) in fresh cycle. As for pregnancy outcomes in FET cycles, no statistically significant differences were observed among the four groups in embryo implantation rate (26.2%), clinical pregnancy rate (63.0%) and abortion rate (11.8%) (all P<0.05)．However, embryo implantation rate, clinical pregnancy rate was higher in PPOS group compared with the other groups. Conclusion Conclusion Compared with the other three ovulation stimulation programme, PPOS might be used as a new alternative for controlled ovulation stimulation protocols.

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Correlation between follicular fluid metabolic markers and oocyte quality in patients with polycystic ovary syndrome

HUANG Jian-lei CHEN Shu-qiang WANG Ming LI Chen-long MU Jing LIU Dan SUN Hui-jun WANG Xiao-hong

2021, 52 (2): 289-294. doi: 10.16098/j.issn.0529-1356.2021.02.020

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Objective To investigate whether the metabolites in follicular fluid can be used as indicators for predicting oocyte quality and embryos early development in patients with polycystic ovary syndrome(PCOS). Methods The study subjects were divided into four groups: lean control (LC) 30 cases, overweight (OW) 13 cases, lean PCOS (LP) 26 cases, and overweight PCOS (OP) 26 cases based on the Chinese criteria for body mass index (BMI) categories. The metabolic variance of follicular fluid from PCOS and controls was performed by liquid chromatography-mass spectrometry (LC-MS), and clinical characteristics, oocyte outcomes and differences of metabolites in follicular fluid of those patients have been compared by ANOVA. Furthermore, Pearson’s correlation analysis was used to explore the correlation between oocyte fertilization rate, top-quality embryos rate and follicular metabolites. Results Compared to the LC group, oocytes retrieved and top-quality embryos rate were significantly increased, while the mature oocytes rate and fertilization rate decreased significantly in the OP group. A total of 236 metabolites were identified and quantified by metabolomics in follicular fluid. Compared with LC and OP groups, 19 metabolites showed statistically significant differences in PCOS group. Additionally, 7 metabolites were significant correlated with the fertilization rate or top-quality embryo rate respectively, most of which were lysophospholipids. Conclusion Several metabolites in the follicular fluid are significantly different between PCOS and healthy women. Among these, lysophospholipids are crucial for oocyte quality and early embryonic development, may serve as potential markers to evaluate the oocyte quality in PCOS patients.

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Protective effect of nuclear factor E2-related factor 2 on oxidative injury in human immortalized epidermal cells

MAO Zhi-rong LIU Fang DU Jie DENG Li GAO Xiao-qing

2021, 52 (2): 225-230. doi: 10.16098/j.issn.0529-1356.2021.02.010

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Objective To investigate the protective effect of nuclear factor E2-related factor 2（Nrf2）on hydrogen peroxide (H2O2)-induced oxidative injury in human immortalized epidermal cells (HaCaT). Methods Nrf2 was overexpressed in HaCaT (Nrf2/HaCaT) through lentiviral transfection, then cell proliferative activity was examined by MTT assay and anti-ki67 immunofluorescent staining. The experiment was divided into Nrf2/HaCaT group and HaCaT group, with five samples in each group. After cells were treated with H2O2 of 200 μmol/L for 24 hours to induce oxidative injury, the cell viability, damage and apoptosis were respectively detected by MTT assay, lactate dehydrogenase (LDH) assay and TUNEL staining. Moreover, the content of the related indicators of oxidative stress, including Nrf2, glutathione（GSH）, superoxide dismutase（SOD）, malondialdehyde（MDA）, reactive oxygen species（ROS）, and the content of the inflammation associated factor, comprising interleukin（IL）-6, tumor necrosis factor（TNF）-α, nuclear factor kappa-B（NF-κB）P65 were checked by ELISA assay and colorimetry assay. Results The proliferative activity of Nrf2/HaCaT was higher than that of HaCaT (P<0.05). When induced by H2O2 for 24 hours, compared with HaCaT, the cell viability increased significantly (P<0.05), and the LDH release and apoptosis rate decreased significantly in Nrf2/HaCaT (P<0.05). The levels of antioxidant Nrf2, GSH, SOD were higher (P<0.05), and the levels of oxidation product ROS, MDA, and inflammatory factor IL-6, TNF-α and NF-κB P65 were lower in supernatant in Nrf2/HaCaT than those in HaCaT (P<0.05). Conclusion Nrf2 overexpression could promote HaCaT proliferation and reduce H2O2-induced oxidative and inflammatory injury.

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Effects of ulinastatin on intestinal mucosal barrier function in sepsis rats and its effect on wnt signal transduction pathway

WANG Yu-hui YE Ba-ning WU Jing LONG Da-li LI Kun SHI Xian-qing

2021, 52 (2): 295-299. doi: 10.16098/j.issn.0529-1356.2021.02.021

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Objective To investigate the intestinal mucosal barrier function protective effect of ulinastatin in sepsis rats and its effect on Wnt/β-catenin signaling pathway. Methods One hundred SD rats were randomly divided into control group, sepsis group, ulinastatin group, XAV939+ulinastatin group and lithium chloride(LiCl)+ulinastatin group. The classical cecal ligation was used to duplicate sepsis model, and the jejunal mucosal injury was evaluated. The levels of inflammatory factors interleukin(IL)-6 and tumor necrosis factor(TNF)-α were detected by ELISA, and the expressions of β-catenin and cyclin D1 were detected by Real-time PCR and Western blotting. We also observed the effect of the Wnt signal pathway blockage by XAV939 or Wnt signal pathway activator by LiCl on ulinastatin protection of intestinal mucosa and proteins related to the Wnt signal pathway. Results The levels of IL-6, TNF-α and intestinal mucosal injury in the sepsis group were significantly higher than those in the ulinastatin group. The mRNA and protein expression levels of β-catenin and cyclin D1 in the sepsis group were significantly higher than those in the control group (P<0.05), After ulinastatin treatment, the expression levels of β-catenin and cyclin D1 mRNA and protein were significantly decreased, and the difference was significant (P<0.05). Compared with the ulinastatin group, combined treatment with XAV939 promoted the protective effect of ulinastatin on the intestinal mucosa of rats, and the protein expression of β-catenin and cyclin D1 was reduced (P<0.05). Combined treatment with LiCl weakened the protective effect of ulinastatin on the intestinal mucosa of rats, and the protein expression of β-catenin and cyclin D1 was increased (P<0.05). Conclusion Ulinastatin may inhibit the Wnt signaling pathway by down-regulating the expression of β-catenin, reduce the expression of inflammatory factors IL-6 and TNF-α, thereby promote repairing the intestinal mucosal barrier function damage.

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Targeted metabolomics analysis of amino acid during rat liver regeneration

YANG Hui XU Cun-shuan

2021, 52 (2): 210-215. doi: 10.16098/j.issn.0529-1356.2021.02.008

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Objective To explore the regulation of amino acid metabolism during rat liver regeneration (LR). Methods Rats were randomly divided into 10 groups with 5 rats in each group, including nine partial hepatectomy (PH) groups and one normal control group. Selective reaction monitoring/multi reaction monitoring(SRM/MRM) was employed in the targeted metabolomics identification of 20 amino acids at 10 time points in rat liver regeneration. The change of amino acid content was analyzed by hierarchical clustering. Results Alanine was up-regulated at 30, 36 and 72 hours; arginine was up-regulated at 6 and 12 hours; aspartic acid was up-regulated at 6, 12 and 36 hours; glutamate was up-regulated at 6, 12, 30, 36, 72 and 120 hours; histidine was up-regulated at 12, 24, 30, 36, 72 and 120 hours; glutamine was up-regulated at 72 hours, and isoleucine was down-regulated at 24 hours. Through hierarchical clustering analysis of amino acids，these amino acids can be grouped into three clusters. Conclusion Many amino acids have changed during the liver regeneration, and throughout the whole process of rat liver regeneration. Changes in amino acids content play an important role in hepatocyte proliferation during the liver regeneration.

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Expression profile of circularRNA during rat liver regeneration and its regulatory effect on cell proliferation

GUO Xue-qiang GUO Jian-lin JIN Wei CHANG Cui-fang XU Cun-shuan CHEN Guang-wen

2021, 52 (2): 216-224. doi: 10.16098/j.issn.0529-1356.2021.02.009

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Objective This study aims to investigate the expression profile and regulatory effect on cell proliferation of circular RNA(circRNA) in rat liver regeneration(LR). Methods CircRNA expression profile during rat LR of 114 rats’regenerating liver which induced by 2/3 partial hepatectomy was detected by high-throughput sequencing. MiRanda and TargetScan were performed to predict their target microRNA(miRNAs) and mRNAs. Gene Ontology(GO) and IPA were used to analyze the physiological activities and signaling pathways they involved. Cytoscape v3.0.2 was used to construct the interaction network. Finally, the candidate key circRNAs were selected by the expression pattern combining with the number and function of target miRNAs. Results 20 878 circRNAs were detected during rat LR, among which 560 of them were differentially expressed, and 126 of them could bind to 117 target miRNAs, which were in turn to regulate 6510 downstream target mRNAs. They were involved in cell proliferation, stress response, substance metabolism and transforming growth factor-β(TGF-β), protein kinase A(PKA), Wnt/beta-catenin signaling pathways. 6 differential expressed circRNAs, including circRNA_03651, circRNA_03653, circRNA_04500, circRNA_05865, circRNA_11274 and circRNA_13559 might play a pivotal role in cell proliferation involved in rat LR by regulating 12 miRNAs and 15 mRNAs. Resultsing they were regarded as the candidate key circRNAs of rat LR. Conclusion 560 circRNAs were differentially expressed in rat LR, among which circRNA_03651, circRNA_03653, circRNA_04500, circRNA_05865, circRNA_11274 and circRNA_13559 might play a crucial role on cell proliferation involved in rat LR via 12 miRNAs-15 mRNAs axis.

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Analysis of body composition of Mongolia Chahar nationality

WU Chao LI Yong-lan

2021, 52 (2): 300-305. doi: 10.16098/j.issn.0529-1356.2021.02.022

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Objective To understand the current situation and characteristics of body composition of Inner Mongolia Chahar tribe. Methods The body composition of 403 adults (161 males and 242 females) in Chahar of Inner Mongolia was measured by bioelectrical impedance analysis. Results The body muscle mass of male and female was the largest, the lower limb muscle mass was medium, and the upper limb muscle mass was the smallest.The results of variance analysis showed that there were statistically significant differences in height, body fat rate, estimated bone mass, visceral fat grade, muscle mass of left and right upper limbs, trunk fat rate and muscle mass among men.The results of correlation analysis showed that the body fat rate increased with age, while the body fat rate, height, estimated bone mass, visceral fat grade, left and right upper limb muscle mass and trunk muscle mass decreased with age.Except for water rate and muscle mass of left and right upper limbs, there were significant differences in other 15 indexes among age groups.The results of correlation analysis showed that trunk fat rate and visceral fat grade increased with age, while height, total muscle mass, estimated bone mass and trunk muscle mass decreased with age.The results of u test showed that except body mass index(BMI), there were statistically significant differences in 17 body composition values between genders in Inner Mongolia Chahar.The total fat percentage and BMI of Inner Mongolia Chahar are in the middle among the seven Mongolian ethnic groups.According to the results of principal component analysis of 11 ethnic groups, the fat content of men and women in Inner Mongolia was higher, and the muscle content was medium. Conclusion The body fat development level of Inner Mongolia Chahar was slightly lower than that of northern Western Mongolian, and slightly higher than that of Eastern Mongolian; the overall development level of body composition is close to that of northern Mongolian, but higher than that of Southern Mongolian.

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Association between the polymorphism of aldosterone synthase gene promoter-344T/C and preeclampsia in Qinghai Province

LI Wen-jia WANG Ru DUAN Qian LI Chang-xing LI Hong-rong LI Jian-hua

2021, 52 (2): 306-310. doi: 10.16098/j.issn.0529-1356.2021.02.023

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Objective To investigate the relationship between preeclampsia (PE) and polymorphism of aldosterone synthase gene (CYP11B2) promoter region-344T/C in Qinghai Province. Methods A total of 120 PE subjects and 155 normal pregnancy subjects were studied. The genotype of CYP11B2 was analyzed by polymerase chain reaction fragment length polymorphism (PCR-RFLP)．The mutation was confirmed by sequencing. Results The frequencies of CYP11B2 TT, CT and CC genotype in the PE group were 43.0%, 45.6%, and 11.4%, and in the control group were 51.0%, 45.1%, and 3.9%, respectively. There was difference in frequency distribution of CYP11B2 genotype between the PE and control groups．The frequency of C allele in the PE group was higher than the control group（χ2=4.354，P<0.05, OR=0.691, 95% CI=0.49-0.98）. Conclusion The CYP11B2-344T/C polymorphism is related to preeclampsia in Qinghai Province, and the C allele may be a susceptibility gene of preeclampsia.

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Whole-mount intramuscular nerve distribution pattern of medial and lateral plantar muscles and its clinical significance

LUO Lin-fen DENG Qun CHEN Li-yun LI Ya-fang YANG Sheng-bo

2021, 52 (2): 264-269. doi: 10.16098/j.issn.0529-1356.2021.02.016

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Objective To reveal the whole-mount distribution pattern of intramuscular nerves in the medial and lateral plantar muscles and to explore its clinical significance. Methods Twenty-four adult cadavers were dissected to remove the medial and lateral groups of the plantar muscles. The distribution pattern of the intramuscular nerves was demonstrated by modified Sihler’s staining. Results The nerve branch for adductor hallucis muscle entered the muscle from the deep surface of the insertion of the muscle, while those nerve branches for abductor hallucis, flexor hallucis brevis, abductor digiti minimi and flexor digiti minimi brevis muscles entered the muscle from the deep side of the origin of the muscle. There were one lunate and one rectangular intramuscular nerve dense regions (INDRs) in the abductor hallucis muscle; two reniform INDRs in the transverse head of the adductor hallucis muscle, one reniform and one rectangular INDRs in the oblique head of the adductor hallucis muscle; there were two rectangle INDRs in the flexor hallucis brevis, abductor digiti minimi and flexor digiti minimi brevis muscles. These five muscles were divided into two neuromuscular compartment, but the percentage position of INDR and the center of INDR on muscle length in each muscle were different. Conclusion These result may provide morphological guidance for surgical operation to avoid nerve injury, the selection and matching of muscle transplantation and the injection of botulinum toxin A to block the spasticity of these muscles.

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Construction of forkhead box J2 gene knockout plasmids by CRISPR/Cas9 and the effects on expression of transforming growth factor-β/Smads and proliferation in hepatocellular carcinoma

QU Ming-juan ZHENG Yu LI Yan-min SONG Yang WANG Lei ZHOU Ju-hua

2021, 52 (2): 231-236. doi: 10.16098/j.issn.0529-1356.2021.02.011

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Objective To construct the clustered regularly interspaced short palindromic repeats/associated protein 9(CRISPR/Cas9) plasmid targeting forkhead box J2(FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β)/Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.

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High protein acetylation/succinylation levels and their correlation with high histone 2AX expression level in breast cancer

GAO Xiu-li YUE Li-ling ZHAO Yue-sheng LIU Li-kun LIU De-shui ZHU Wen-bin ZHOU Li

2021, 52 (2): 244-250. doi: 10.16098/j.issn.0529-1356.2021.02.013

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Objective To explore the protein acetylation/succinylation and histone 2AX(H2AX) expression levels in breast cancer, as well as their correlation. Methods By Western blotting and RT-PCR methods to detect the protein modification and H2AX expression levels in 11 breast cancer tissues and cells, as well as to explore the common regulation way of protein acetylation and succinylation by treatment of histone deacetylase inhibitors; To study the relationship between H2AX expression level and protein modification level through the construction and over-expression of indicated plasmids. Results Compared with the adjacent normal tissues, there existed an increase protein acetylation/succinylation levels in breast cancer tissues, and the protein acetylation and succinylation were both regulated by histone deacetylase(HDAC) members. The H2AX mRNA and protein expression levels were increased both in breast cancer cell and tissues, its expression level and the expression and modification level of represented protein nucleophosmin 1(NPM1) showed a positive correlation. Conclusion The breast cancer possesses a characteristic of high protein acetylation/succinylation levels and high H2AX expression level, the H2AX expression level and the modification level of partial proteins in breast cancer have a positive correlation.

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Co-expression of plasmid-based protein kinase B1-specific small interfering RNA and P53 synergistically inhibits proliferation, migration and invasion of hepatocellular carcinoma cells

CHEN Xiao-long WANG Ya-feng HUANG Ping

2021, 52 (2): 251-257. doi: 10.16098/j.issn.0529-1356.2021.02.014

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Objective To investigate the effect of the dual expression plasmid of protein kinase B1(Akt1)-specific siRNA and P53 on the proliferation, migration, invasion and apoptosis of hepatocellular carcinoma (HCC) cells. Methods We constructed a dual expression plasmid that co-expressed Akt1-specific siRNA and wild-type p53 gene (pSi-Akt1-P53).The dual expression plasmid pSi-Akt1-P53 was transfected into HepG2 cells of HCC,The expression of Akt1 and P53 was detected by Real-time PCR and Western blotting. Then, the dual expression plasmid was transfected into HepG2 cells, sh-Akt1 plasmid and P53 plasmid were used as control. The effects of the dual plasmid on the proliferation, migration, invasion and apoptosis of HepG2 cells were detected by CCK-8 and 5-ethynyl-2’-deoxyuridine(EdU) experiments, Wound scratch experiment, Transwell chamber experiment and flow cytometry,respectively. Results After the dual plasmid was transfected into HepG2 cells, the expression of Akt1 protein was significantly reduced and the expression of P53 protein was significantly increased in HepG2 cells. Compared with the shAkt1 and P53 plasmids, the dual expression plasmid pSi-Akt1-P53 significantly inhibited the proliferation、migration and invasion of HepG2 cells and significantly increased the apoptosis of HepG2 cells. Conclusion The dual expression plasmid pSi-Akt1-P53 can synergistically inhibit the proliferation, migration and invasion of HepG2 cells, significantly increased the apoptosis of HepG2 cells.

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Hypoxia promotes lung adenocarcinoma A549 cells migration by upregulating acetyl-CoA carboxylase 1

JIN Jia-hao ZHAO Bao-sheng LIU Dan-hui LIU Y-zhen

2021, 52 (2): 258-263. doi: 10.16098/j.issn.0529-1356.2021.02.015

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Objective To investigate the mechanism of hypoxia to promote human lung adenocarcinoma A549 cells migration through acetyl-CoA carboxylase 1 (ACC1). Methods Lung adenocarcinoma A549 cells were treated with hypoxia (5% O₂). Transwell migration assay was used to detect cell migration ability. Western blotting was used to detect ACC1 expression and epithelial-mesenchymal transition (EMT) related protein expression. Results Compared with the normoxia (control group), hypoxia treatment promoted the migration of A549 cells (P<0.01), ACC1 expression was up-regulated after hypoxia treatment (P<0.01), and vimentin expression was detected to increase significantly (P<0.05), E-cadherin expression decreased (P<0.01); Compared with the control group, migration of A549 cells was inhibited (P<0.05), vimentin expression was down-regulated (P<0.05), and E-cadherin expression increased after knocking down ACC1(P<0.01). After ACC1 was knocked down, the differences between the numbers of migration of A549 cells under normoxia and 5% O2 conditions and the expressions of vimentin and E-cadherin were not statistically significant (P>0.05). After linoleic acid (LA) supplementation, the hypoxia-induced migration promotion of A549 cells was restored. Conclusion Hypoxia can promote the migration and EMT transformation of lung adenocarcinoma A549 cells by up-regulating the expression of ACC1.

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Lower expression of dynein axonemal intermediate china 1 in lung adenocarcinoma and inhibits the invasion of lung adenocarcinoma cells

ZHANG Lin WANG Xue-ting WANG Xiao-dong WANG Yu LI Chun-tao YIN Chong-gao LI Hong-Li

2021, 52 (2): 237-243. doi: 10.16098/j.issn.0529-1356.2021.02.012

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Objective To investigate the expression of dynein axonemal intermediate chain 1(DNAI1) in lung adenocarcinoma(LUAD) and its influence on invasive ability of lung adenocarcinoma. Methods Microarray gene chip analysis was used to screen different expression genes in lung adenocarcinoma(3 samples) and adjacent normal tissues(3 samples); Heatmap and volcano plot were performed demonstrate the mRNA expression and distribution after screening; DAVID database used for Gene Ontology(GO) and Kyoto Encyclopedia of Genes of Genomes(KEGG) analysis; STRING database and Cytoscape 3.6.1 software for protein-protein interaction (PPI) analysis and screening of Hub genes; Objective genes were selected based on the differential expression of each Hub gene in lung adenocarcinoma in DEGs and Ualcan database ; Real-time PCR and Western blotting were used to detect the expression of DNAI1 in BEAS-2B, H1299 and A549; observe the morphological changes after DNAI1 overexpression; Transwell invasion assay was used to detect the change of invasion ability of A549 cells after DNAI1 overexpression. Results The microarray result showed that there were 86 up-regulated genes and 396 down-regulated genes; different genes were involved in the RNA polymerase Ⅱ promoter positive regulation of transcription, apoptosis process of negative regulation, protein binding, and other functions, widely distributed within the cell, and associated with the metabolic pathway, cancer and other signal pathways were closely related; DEGs database and Ualcan database showed that DNAI1 was the most downregulated among Hub genes in LUAD; the result of Real-time PCR and Western blotting showed that DNAI1 had lower expression in H1299 and A549 compared with BEAS-2B; after DNAI1 overexpression, A549 cells became round and a few shed off; invasion assay showed that the invasion ability of A549 cells was significantly reduced. Conclusion DNAI1 has a lower expression and inhibits the ability of invasion in LUAD, and this study can provide a potential molecular target and provide a theoretical basis for targeted therapy of LUAD.

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Expressions of iron transport related proteins in the spinal cord of amyotrophic lateral sclerosis transgenic mice

ZHANG Ya-wen GAO Ying SUN Han-cong ZHANG Hao-yun WANG Feng-bin

2021, 52 (2): 161-167. doi: 10.16098/j.issn.0529-1356.2021.02.001

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Objective To investigate the relationship between the expressions of iron transport related proteins and the dysregulation of iron homeostasis in the spinal cord of amyotrophic lateral sclerosis(ALS) transgenic mice. Methods The hSOD1G93A transgenic mice (ALS mice) and littermate wild-type mice (WT mice) were selected to separate the spinal cord at day 70, day 95, and day 122 after birth, 9 mice per time point and per group. Western blotting was used to detect the expressions of iron transporter divalent metal transporter-1 (DMT1), ferroportin 1 (FPN1) and regulatory protein iron regulatory protein 1(IRP1) in the spinal cord. Double immunofluorescence labeling was used to detect the co-localization of cells in the ventral horn of lumbar spinal cord. Results Western blotting results showed that compared with WT mice the expressions of DMT1 protein were down-regulated with the disease progression from day 70 to day 122 (P<0.05, P<0.01); FPN1 protein was transiently up-regulated at day 70 (P<0.05), and decline expressions were observed at day 95 and day 122 (P<0.01); IRP1 was down-regulated at day 95 and day 122 (P<0.01). Double immunofluorescence labeling revealed that at day 70, DMT1 co-expressed mainly with β-tubulin Ⅲ both in WT and ALS mice lumbar spinal cord. Compared with the WT group, the DMT1 immunoreactivity in the neurons of the ventral horn lumbar spinal cord of ALS mice was elevated at day 95, while the FPN1 fluorescence intensity was weak. With the disease progression, the co-localization expression of DMT1, FPN1 with reactive glial cells increased. With the disease progresses, the expression of IRP1 decreased. Conclusion With the progression of ALS, iron influx increases and iron outflux decreases in neurons at the early-symptomatic stage of ALS, the activity of iron transport in reactive glial cells is enhanced, which participates in local iron homeostasis imbalance and progressive loss of motor neurons in ventral horn of spinal cord. Decreased expression of IRP1 partly participates in the regulation of local iron metabolism.

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Quality control analysis of brain tissue samples from Shanghai Brain Bank

LI Feng-jiao XIAN Wei-wei BU Shuo-lei CAO Jing-li WU Jin-song LI Wen-sheng YOU Lin-ya

2021, 52 (2): 168-174. doi: 10.16098/j.issn.0529-1356.2021.02.002

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Objective To evaluate the feasibility of using brain tissues from Shanghai Brain Bank for the applications on biological research through the analysis of pH value of cerebrospinal fluids, RNA integrity number (RIN), transcriptome, proteome and morphology of brain tissues. Methods The pH value of fresh cerebrospinal fluid was detected by pH test paper; the RNA integrity of cryopreserved brain tissues was examined by Agilent RNA 6000 Nano chip and Agilent 2100 bioanalyzer; the transcriptome sequencing of superior temporal gyrus or caudate was performed using BGIseq-500 sequencer; the proteome of cryopreserved brain tissues was analyzed by Q Exactive HF mass spectrometer; at last, the morphology of the superior temporal gyrus was observed by HE staining. Results The pH value of the cerebrospinal fluid on average was about 6.5. The RIN values of more than 65% of brain tissues were more than 6, indicating good RNA quality. The clean reads ratio after transcriptome sequencing filtering was basically above 80%, indicating that the quality of sequencing library was high. The mass spectrometry analysis of frozen brain samples yielded more than 4000 protein groups and 30 000 peptides, indicating high quality of proteomic data. The morphology of brain tissues was relatively normal, with clearly visible neurons. Conclusion The quality of RNA and protein of brain tissues from Shanghai Brain Bank meets the basic needs for molecular and biological research, and the fixed brain samples can be used for morphological observations.

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Dynamic expression of putative receptor protein related to the angiotensin type 1 receptor in postnatal mouse cochlear hair cells and spiral ganglion neurons

YIN Hai-yan WANG Ze-yu HAO Chang-jiang ZHANG Hui WANG Feng-qin LI Xiu-guo CHEN Jing

2021, 52 (2): 175-181. doi: 10.16098/j.issn.0529-1356.2021.02.003

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Objective To study the temporal and spatial expressions of G protein-coupled receptor, putative receptor protein related to angiotensin type 1 receptor (APJ), in mammal cochlea postnatal development. Methods The cochlear tissues of each group 11 C57BL/6 mice at postnatal day 7 (P7), P14, P28 and postnatal month 2(P2M) were taken out under a stereo microscope. Real-time PCR, Western blotting and immunofluorescent staining were used to detect the expressions of APJ in hair cells and spiral ganglion neurons. Results The expression pattern of APJ in cochleae showed an upward trend during the period from P7 to P2M. The temporal expressions of APJ in hair cells and spiral ganglion neurons increased obviously at P14 and P2M. The spatial expression patterns of APJ in hair cells and spiral ganglion neurons followed a declined gradient from base turns to apex turns at P14. Conclusion APJ expression exhibits a specific spatial and temporal pattern during mouse cochlea postnatal development, and may play a role in cochleae maturation and hearing formation.

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Expression and role of Huntingtin-associated protein 1 during valproate induced neural stem cells differentiation

WANG Shanshan LI Wen HE Hui QIN Jian-bing TIAN Mei-ling ZHAO He-yan CHENG Xiang JIN Guo-hua

2021, 52 (2): 182-188. doi: 10.16098/j.issn.0529-1356.2021.02.004

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Objective To investigate the expression and role of Huntingtin-associated protein-1 (HAP-1) in the process of valproate acid (VPA) inducing neural stem cells (NSCs) into neurons. Methods The hippocampus NSCs of SD rats were isolated and cultured, Real-time PCR and Western blotting were used to detect HAP-1 mRNA and protein expression at day 0, day 1, day 3 and day 5 during the induction of VPA on NSCs differentiation into neurons; Real-time PCR was used to detect the expression level of HAP-1 mRNA in multiple tissues of adult SD rats, as well as NSCs, neurons and astrocytes. After applying small interfering RNA technology to down-regulate the expression of HAP-1 mRNA in NSCs, Real-time PCR was used to detect the mRNA expression levels of neuron-specific molecules stathmin-2 (Stmn-2), neuronal differentiation-1 (Neurod-1), microtubule-associated protein-2 (Map-2) and synapsin-1 (Syn-1), and Western blotting was used to detect the protein expression levels of neuron-specific marker β-tubulinⅢ（Tuj-1）. Immunofluorescence was used to detect the proportion of NSCs differentiated into Tuj-1 positive neurons, and to observe the development of neurons. Results At day 1 and day 3 after VPA treatment, the expression of HAP-1 mRNA and protein in the VPA group was significantly up-regulated; HAP-1 mRNA was predominantly expressed in the hippocampus, and its expression was higher in neurons, followed by NSCs, and minimally in astrocytes. After down-regulating HAP-1 with small interference technology, the proportion of NSCs differentiated into Tuj-1 positive neurons reduced, and neuron development became worse. Conclusion VPA may promote the differentiation of NSCs into neurons by up-regulating HAP-1 expression.

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Effects of volatile oil from Acori Graminei Rhizoma on glial fibrillary acidic protein and immediate early genes expressions in the basal lateral amygdala of the inflammatory pain rats

LI Shi-qi YANG Cui-zhu LIU Hong-qing ZHANG Chun-mei TIAN Su-min LIU Jing MA Yu-xin LI Guo-ying

2021, 52 (2): 189-195. doi: 10.16098/j.issn.0529-1356.2021.02.005

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Objective To construct a rat model of inflammatory pain by injecting complete Freund’adjuvant (CFA) to study effects of volatile oil of Acori Graminei Rhizoma on the expression of glial fibrillary acidic protein (GFAP) and immediate early gene c-fos in the basal lateral amygdale (BLA) of the inflammatory pain rats. Methods Thirty-six adult male SD rats were randomly divided into 6 groups: control group, sham group, CFA group, CFA+5 g/(kg · day) volatile oil of Acori Graminei Rhizoma group, CFA+10 g/(kg · day) volatile oil of Acori Graminei Rhizoma group, CFA+ 20 g/(kg · day) volatile oil of Acori Graminei Rhizoma group, six rats in each group were taken gavage for 21 days. Immunofluorescence and Western blotting methods were used to detect the expressions of GFAP and c-fos in the BLA of all rats. Results Immunofluorescence and Western blotting results showed that compared with the control group, the positive expression of GFAP and c-fos in the BLA of the CFA rats were significantly increased (P<0.01). After treatment of the volatile oil from Acori Graminei Rhizoma, the positive expressions of GFAP and c-fos were reduced compared to the CFA group, as well as the expression levels were decreased in the dose-dependent manner (P<0.01). Compared with the low dose group, the positive expression of GFAP and c-fos of high dose group were decreased significantly (P<0.01). Conclusion The volatile oil fraction from Acori Graminei Rhizoma could reduce the expressions of GFAP and c-fos the BLA of CFA-induced chronic inflammatory pain model rats.

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Effect and mechanism of picroside Ⅱ on p38 mitogen activated protein kinase signal transduction pathway after cerebral ischemia/reperfusion in rats

YU Zhu-qin WANG Guan-xi WANG Xiao-lu WANG Yue WANG Ting-ting

2021, 52 (2): 196-204. doi: 10.16098/j.issn.0529-1356.2021.02.006

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Objective To explore the neuroprotective effect and mechanism of picroside Ⅱ on p38 mitogen activated protein kinase（p38 MAPK）signal transduction pathway after cerebral ischemia/reperfusion injury in rats. Methods A total of 150 healthy male Wistar rats were subject to establish middle cerebral artery occlusion/reperfusion (MCAO/R) models by inserting a monofilament thread. All rats were randomly divided into sham group, model group, picroside (Picr) group, anisomycin (Anis，agonist of p38 MAPK) group, Anis+Picr group, SB203580 (SB, inhibitor of p38 MAPK) group and SB+Picr group. The neurobehavioral function was evaluated by modified neurological severity score points (mNSS) test. The structure of neuron was observed using HE staining. The apoptotic cells were counted using TUNEL assay. The expression of phosphorylated p38 MAPK (p-p38 MAPK) in cortex was determined using the immunohistochemistry. And the expressions of p-p38 MAPK, phosphorylated MAPK activated protein kinase-2 (p-MK2), phosphorylated cytoplasm phospholipase A2 (p-cPLA2), interleukin-6 (IL-6) and tumor necrotic factor α (TNF-α) were determined by Western blotting. Results No neurological behavioral malfunction was found in sham group. In model group, the damage of neuron was worsened, while the neurobehavioral function score, apoptotic cell index and the expressions of p-p38 MAPK, p-MK2, p-cPLA2，IL-6 and TNF-α increased significantly than those in control group. No significant difference was found in TNF-α. In Picr group, SB group and SB+Picr group, the damage of neuron was lighter, the neurological behavioral function was improved, the number of apoptotic cells and the expressions of p-p38 MAPK, p-MK2, p-cPLA2 and IL-6 decreased significantly than those in model group. In Anis group and Anis+Picr group, the damage was worsen, the cerebral infarction was larger, and the expressions of p-p38 MAPK, p-MK2, p-cPLA2 and IL-6 increased significantly than those in control group. Conclusion Picroside Ⅱ can protect the neuron from the apoptosis and inflammation reaction after MCAO/R by inhibiting p38 MAPK signal transduction pathway in rats.

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Therapeutic effect and mechanism of hydrogel-encapsulated human umbilical cord mesenchymal stem cells on traumatic brain injury in rats

ZHANG Yu XUE Qian SONG Xiao-yu YIN Wei-dong ZOU Yu-an

2021, 52 (2): 205-209. doi: 10.16098/j.issn.0529-1356.2021.02.007

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Objective To investigate the therapeutic effect of hydroge-encapsulated human umbilical cord mesenchymal stem cells (hUC-MSCs) on traumatic brain injury in rats and its related mechanism. Methods SD rat models of traumatic brain injury were constructed, which were divided into control group, chitosan group, stem cell group and combined treatment group. Results During the treatment, there was no significant difference in mNSS score between the control group and the chitosan group (P>0.05). On the 15th, 22nd, 29th and 36th day, the mNSS score of the combined treatment group decreased most significantly than that of the control group, followed by the stem cell group(P<0.05). Compared with the control group, the escape latency of the combined treatment group decreased most significantly, followed by the stem cell group (P<0.05). The number of beta-Ⅲ tubulin-positive cells in the brain tissues of rats in the treatment group was significantly higher than that in the control group. Compared with the control group, there was no significant change in the expression of neuron-specific enolase (NSE), neuron-specific nuclear protein (NeuN), microtubule associated protein 2 (MAP2), brain-derived neurotrophic factor (BDNF) and B cell lymphoma-2 (Bcl-2) in the chitosan group (P>0.05), and the expression of related proteins in the stem cell group and the combined treatment group increased significantly. The expression level of the related protein in the combined treatment group was the highest, followed by stem cell group, the lowest in the control group and chitosan group (P<0.05). Conclusion Compared with stem cell transplantation alone, hydrogel-encapsulated hUC-MSCs transplantation can improve the motor and learning and memory abilities of rats with traumatic brain injury more effectively.

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