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    05 April 2023, Volume 43 Issue 4
    Invited Reviews: Basic Research of Reproduction
    Advances in research on RNA N-6-methyladenosine(m6A) modification related enzymes in mammalian spermatogenesis
    ZHANG Qiang, SU Wenhui
    2023, 43(4):  524-531.  doi:10.16352/j.issn.1001-6325.2023.04.0524
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    N-6-methyladenosine(m6A) has been well-known to be a type of RNA epigenetic modification over the past decade. The m6A modification enzymes include “Writers” (METTL3/14/WTAP, etc.),“Erasers” (FTO and ALKBH5) and “Readers” (YTHDC1/2,YTHDF1/2/3, etc.), which mediate RNA methylation, demethylation, and recognition binding via a synergistic regulatory system, thereby regulating the fate of RNA after transcription. Spermatogenesis is an essential process of sexual maturation and maintenance of fertility in male mammals, involving the proliferation, and differentiation of Sertoli cells, Leydig cells and spermatogonia, and maintenance of spermatogenic microenvironment. More recently, there are some researches showed that the m6A regulatory system was involved in mammalian spermatogenesis. Abnormal m6A modification and imbalances in the m6A regulatory system can lead to abnormal testicular development, abnormal spermatogenesis and male infertility. This review summarizes the function of m6A modification enzymes in spermatogenesis, and further analyzes the role of m6A differentially modified transcripts in normal spermatogenesis, which has a great significance for understanding mammalian spermatogenesis and decoding the molecular mechanism of clinical spermatogenesis disorders.
    Advances in the effect of NC1 domain of type Ⅳ collagen on spermatogenesis and blood-testis barrier
    JIANG Shuyi, SU Wenhui
    2023, 43(4):  532-537.  doi:10.16352/j.issn.1001-6325.2023.04.0532
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    In mammalian testis, the blood-testis barrier (BTB), located near the basement membrane of seminiferous epithelium, is essential for spermatogenesis. BTB is formed by the joining of adjacent Sertoli cells and divides the seminiferous epithelium into basal compartment near the basement membrane and apical compartment near the lumen of spermatogenic tubules. The barrier provides an immune defense mechanism for spermatogenesis. Type Ⅳ collagen is an important component of basement membrane. In rodent testis, type Ⅳ collagen is mainly composed of three α3 chains in a triple helix structure. The C-terminal noncollagenous domain (NC1 domain) of type Ⅳ collagen α3 chain of rat testis can be hydrolyzed into a 28 ku protein fragment called NC1 domain. NC1 domain as a basement membrane peptide is involved in spermatogenesis and sustains BTB function. This article reviews recent research results in order to identify the role of type Ⅳ collagen NC1 domain in testis and underlying mechanisms, mainly focuses on spermatogenesis and BTB.
    In vitro follicle activation as a strategy for assisted reproduction in patients with premature ovarian insufficiency: research advances
    LIU Zhan'ao, CHEN Chen
    2023, 43(4):  538-546.  doi:10.16352/j.issn.1001-6325.2023.04.0538
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    Premature ovarian insufficiency (POI) refers to ovarian dysfunction in women before the age of 40 years, and its pathological basis is the depletion or difficult activation of primordial follicle. POI patients have a low natural conception rate and poor effect of ovulation induction therapy. At present, there is no effective method to restore ovarian function. In vitro activation (IVA) for follicles refers to surgical removal of part of the ovarian tissue, followed by drug or physical intervention in vitro, to activate the patient's remaining follicles. Finally, the tissue is rafted back to the same ovary so that it can develop to the stage of responding to ovulation induction. In conjunction with the above procedure, ovulation induction can obtain mature follicles for in vitro fertilization and embryo transfer. IVA technology is an innovative therapeutic option for POI, and provides a new direction for the treatment of POI patients. This article summarizes the principle of IVA, related signaling pathways and drug targets in each pathway, and reviews the application and optimization of IVA protocols in POI patients, so as to provide a basis for the diagnosis and treatment of patients with POI.
    Advances in molecular mechanisms of intrauterine adhesions
    LU Yimeng, TAN Jichun
    2023, 43(4):  547-553.  doi:10.16352/j.issn.1001-6325.2023.04.0547
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    Intrauterine adhesions (IUA) is a refractory disease affecting female reproductive health. With the development of medicine, the etiology of IUA has been further studied. At present, the high risk factors that can lead to IUA include: curettage after pregnancy, post-caesarean section, trophoblastocyte disease curettage, postpartum hemorrhage curettage, diagnostic curettage, hysteroscopic surgery, intrauterine device placement, reproductive tract tuberculosis infection, Müllerian duct malformation, etc. Endometrial fibrosis is a main pathological feature. After various signals stimulate different molecular signaling pathways, various growth factors and cytokines interact with each other in vivo to promote the development of fibrosis, which ultimately lead to the occurrence of IUA. At present the current clinical treatment and prevention of IUA is not ideal, especially for moderate and severe IUA. Therefore, the research of the pathogenesis may support the targeted therapy for IUA. This paper aims to review the recent progress in the etiology and molecular mechanism of IUA.
    Original Articles
    Construction of mouse liver aging model by hepatocyte organoid
    LU Yan, ZHOU Yabo, CHEN Jie
    2023, 43(4):  554-559.  doi:10.16352/j.issn.1001-6325.2023.04.0554
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    Objective Construction of mouse liver senescence model induced rapidly in vitro by 3D organoid culture. Methods Mouse hepatocytes were collected by two-step perfusion method and cultured in 3D. Organoids were preliminarily identified by morphology, qPCR and immunofluorescence. The function of hepatocyte organoid was evaluated by their uptake of LDL. The hepatocyte organoids were treated with oleic acid, and their clonal growth, senescence related secretory phenotype, LDL uptake and ROS level of liver cells were detected. The hepatocytes were further treated with N-acetylcysteine (NAC) to detect the above phenotypic and functional changes. Results A large number of active primary mouse hepatocytes were obtained by two-step perfusion method. The hepatocyte organoid with self-reproduction ability was collected by 3D culture in the matrix gel. The expression of liver marker genes in hepatocyte organoid and primary hepatocytes was shown by qPCR detection. The immunofluorescence results showed that it highly expressed ALB protein. Moreover, hepatocyte organoid had normal uptake function of LDL. After treatment with oleic acid, the growth of hepatocyte organoid was slowed down, the expression of senescence related secretory phenotype genes was up-regulated(P<0.001), the uptake of LDL was reduced(P<0.001), and ROS was significantly increased(P<0.001). The above phenotypes were alleviated by NAC treatment(P<0.01 or P<0.001). Conclusions Oleic acid treatment can successfully construct the aging model of mouse liver in vitro. Adding NAC can clear ROS and alleviate the occurrence of aging.
    Effect of Pabpc6 knockout on spermatogenesis of male mice
    TANG Jielin, LIU Jun, ZOU Dingfeng, MIAO Shiying, SONG Wei, LI Kai
    2023, 43(4):  560-567.  doi:10.16352/j.issn.1001-6325.2023.04.0560
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    Objective To investigate the role of cytoplasmic poly (A) binding protein-6 (PABPC6) in spermatogenesis of male mice. Methods The Pabpc6 in C57BL/6J mice was knocked out by CRISPR/Cas9 targeting, and the Pabpc6 was identified by PCR. Three types of mice with different genotypes of Pabpc6+/+, Pabpc6+/- and Pabpc6-/- were obtained by mating and breeding. Testis was weighed;Morphological observation and sperm counting were carried out. The mRNA and protein expression levels of Pabpc6 in different tissues were detected by real-time quantitative PCR and Western blot. Immunofluorescence and hematoxylin-eosin staining were used to observe the location of the gene and the morphological changes in the convoluted spermatic tubule of testis. Results The Pabpc6 knockout mice were successfully identified. Pabpc6 was highly expressed in testicular tissue(P<0.001). Immunofluorescence showed that Pabpc6 was mainly expressed in spermatocyte at early sperm stage. Compared with wild-type mice, knockout mice showed no significant differences in testicular shape, sperm morphology, sperm count and the morphology of convoluted spermatic ducts. Conclusions Pabpc6 is found to be highly expressed in the testis tissue of mice. Pabpc6 has no obvious effect on spermatogenesis of male mice after knockout of Pabpc6. The results indicate that the protein encoded by Pabpc6 is not necessary for spermatogenesis of mice.
    Stable knockout of Mcart-1 down-regulates the proliferation and oxidative phosphorylation of RAW264.7 macrophages
    ZHAO Yongjing, QIU Jiaxing, ZHANG Diya, GUO Hongjiang, WANG Yucheng, JU Rui, GUO Lei
    2023, 43(4):  568-575.  doi:10.16352/j.issn.1001-6325.2023.04.0568
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    Objective To construct a RAW264.7 macrophage cell strain with stable knockout of Mcart-1 by using CRISPR/Cas9 gene editing technique, and to detect its biological function. Methods Cas9 and sgRNA lentivirus were used to infect RAW264.7 macrophages in two steps. Positive cells were screened with puromycin and hygromycin, and single cells were plated by flow cytometry to obtain monoclonal cells; Expression of Cas9 and Mcart-1 was detected by qPCR and Western blot, and the mutation site was confirmed by sequence analysis. Cells number counting and carboxyfluorescein diacetate succinimdyl ester (CFSE) staining were used to detect cell proliferation; ATP detection kit was used to measure total ATP content in cells; Seahorse bioenergy analyzer was used to detect cell oxygen consumption rate(OCR) and glycolysis rate. Results Successfully constructed RAW264.7 cell strain with Mcart-1 stably knocked out, denoted as Mcart-1-/--RAW264.7; A frameshift mutation occurred in Mcart-1 gene in this cell strain; Compared with the wild-type RAW264.7 cell line (RAW264.7), the OCR increased and the extracellular acidification rate(ECAR)of Mcart-1-/--RAW264.7 cells decreased; The total ATP content in Mcart-1 knocked out cells decreased(P<0.05),mitochondrial ATP production rate decreased (P<0.01), while glycolytic ATP production rate increased (P<0.01); The proliferation activity of RAW264.7 cells decreased after Mcart-1 was knocked out(P<0.001). Conclusions The proliferation activity and oxidative phosphorylation level of RAW264.7 cells were both down-regulated after the Mcart-1 was stably knocked out. This cell strain is an important tool for exploring the function of cells in the tumor microenvironment.
    Association of ARHGEF7 genetic variants with intracranial aneurysm
    WU Yiyi, ZHANG Mei, YANG Yunyun, LI Yaqiang, LI Jing, HE Jiale, ZHANG Weili
    2023, 43(4):  576-582.  doi:10.16352/j.issn.1001-6325.2023.04.0576
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    Objective To identify the relationship between Rho guanine nucleotide exchange factor 7 (ARHGEF7) genetic variants and the formation and rupture risk of intracranial aneurysms. Methods Ten variants of the ARHGEF7 were detected in a case-control study which included 121 patients with intracranial aneurysms (including 41 unruptured and 80 ruptured patients) and 155 healthy controls. The relationship between variants and occurrence and rupture risk of intracranial aneurysms was examined by multivariate logistic regression model. Results After adjustment for age, sex, and traditional cardiovascular risk factors, the rs4145274GA genotype was associated with a decreased rupture risk of intracranial aneurysms as compared with rs4145274GG genotype, and odds ratio was 0.24[95% confidence interval (CI):0.09~0.69, P<0.05]. The rs1555751CT genotype was also associated with a decreased rupture risk of intracranial aneurysms as compared with rs1555751CC genotype, and odds ratio was 0.17(95% CI: 0.05~0.63, P<0.05). The combined effect rs4145274 and rs1555751 suggested that the patients with an increased genetic risk score had a lower rupture risk of intracranial aneurysms, and the odds ratio was 0.14(95% CI: 0.04~0.55, P<0.05). Conclusions The study indicates that ARHGEF7 genetic variants potentially serve as potential genetic markers for the risk evaluation of ruptured intracranial aneurysms.
    Fluorescence imaging of arterial blood spiral laminar flow of rats
    QI Xi, LI Bei, LI Hongyi, LI Yansong, ZHAO Lihao, HAO You, YU Xue
    2023, 43(4):  583-587.  doi:10.16352/j.issn.1001-6325.2023.04.0583
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    Objective To estatblish a real-time fluorescence imaging method for observing arterial blood spiral laminar flow. Methods Sprague Dawley rats were randomly divided into vein angiography group (femoral vein injection, 1 mL/kg) and low-dose arteriography group (abdominal aortic injection, 0.1 mL/kg). The low-dose arteriography group was further divided into fluorescein sodium group, rhodamine B group, FITC-dextran group and quantum dot group (n=7 for each group). The dynamic images were captured with high-speed camera. The fluorescence region was calibrated by image threshold segmentation algorithm and morphological post-processing algorithm. The axial velocity and tangential velocity of helical blood flow in rat femoral artery were measured, and the screw pitch and shear force were calculated. Results Compared with the vein angiography group, the low-dose arteriography group clearly showed the clockwise spiral laminar flow in the femoral artery of rats. In the FITC-dextran group, the axial velocity of blood spiral laminar flow was (198.4±112.7)mm/s, and the tangential velocity was(3.3±0.8)mm/s.The screw pitch was (126.1±76.3)mm, and the shear force was (9.5±5.6)Pa. The injections were repeated three times for each rat, and there was no significant difference among each injection. Conclusions Real-time fluorescence imaging can be used to observe arterial blood spiral laminar flow in vivo and to quantitatively analyze the hemodynamic characteristics of spiral laminar flow.
    Circadian clock protein CRY1 inhibits Ang Ⅱ-induced phenotypic transformation of mouse aorta smooth muscular cells
    YUE Xiuqing, SONG Qitai, LIU Chaoli, LI Sangrou, YANG Fan, CHEN Muhu, ZHONG Wu
    2023, 43(4):  588-595.  doi:10.16352/j.issn.1001-6325.2023.04.0588
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    Objective To investigate the effect of circadian clock protein cryptochrome 1 (CRY1) on angiotensin Ⅱ(Ang Ⅱ)-induced phenotypic transformation of mouse aorta smooth muscular cells. Methods VSMCs were divided into control group and experimental group. VSMCs were treated with different concentrations of Ang Ⅱ for various time, α-smooth muscle actin (α-SMA), osteopontin (OPN) and CRY1 expressions in VSMCs were detected via RT-qPCR and Western blot. VSMCs were transfected with knockdown vector siCRY1 and siYAP1 or overexpression vector, and the corresponding gene expression was detected with RT-PCR and Western blot. VSMCs were divided into 4 groups: Control group, Ang Ⅱ group, Ang Ⅱ+siCRY1 group and Ang Ⅱ+pcDNA-CRY1 group. siCRY1, siYAP1, pcDNA-CRY1 were transfected into VSMCs induced by Ang Ⅱ for observation on α-SMA, CRY1, OPN and YAP1 at mRNA and protein levels detected by RT-PCR and Western blot. CCK-8 method were used to measure proliferation capacity. Results VSMCs were treated with different concentrations of Ang Ⅱ for various time, the expression of CRY1 was decreased in a model of Ang Ⅱ-induced VSMC phenotypic transformation, which contractile markers down-regulated and synthetic markers up-regulated at mRNA and protein levels. After VSMCs were transfected with siCRY1, siYAP1 and pcDNA-CRY1, the expression of CRY1 decreased in siCRY1 group and increased in pcDNA-CRY1 group, while the expression of YAP1 decreased in siYAP1 group and increased in siCRY1 group.Knockdown of CRY1 could stimulate the transformation of VSMCs from contractile phenotype into synthetic phenotypes by further activating expression of synthetic markers and CRY1(P<0.05), inhibiting expression of contractile markers and YAP1(P<0.05), the proliferation of VSMCs increased significantly(P<0.05). While overexpressed CRY1 could block the phenotypic transformation, the expression of synthetic markers and CRY1 was reduced and contractile markers and YAP1 was increased both at mRNA and protein levels(P<0.05), the proliferation of VSMCs inhibited(P<0.05). Conclusions CRY1 may inhibit switch of mouse VSMCs phenotypes induced by Ang Ⅱ and inhibit VSMCs proliferation, which is potentially related to the Hippo-YAP signaling pathway.
    Influencing factors and the clinical significance of tumorigenesis rate in PDX model of colorectal cancer
    ZHANG Yanping, YAO Liuxu, DING Qiannan, HUANG Zeyong, LI Yuhong, HUANG Suqin
    2023, 43(4):  596-602.  doi:10.16352/j.issn.1001-6325.2023.04.0596
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    Objective To establish a patient derived xenograft (PDX) model of tumor tissue from patients with colorectal cancer (CRC) and to identify the factors affecting the tumorigenesis rate of PDX model, as well as to conduct a preliminary chemotherapy. Methods From November 2019 to October 2020, CRC patients undergoing elective surgery in Shaoxing People's Hospital were selected. The tumor tissue obtained from surgical operation was inoculated to the right lumbar back of NSG mice to establish a PDX model, which was subcultured to F3 generation, and the influencing factors of the tumor formation rate of PDX model were analyzed. Chemotherapy drugs include 5-fluorouracil, oxaliplatin and anesthetic propofol. Results A total of 60 patients with CRC were included in this study and 37 samples from patients had PDX tumor formation in mice with a tumorigenesis rate of 62%; The average tumorigenesis time was (34±12)d; Primary tumor malignant degree (tumor stage and degree of cell differentiation), preoperative carcinoembryonic antigen (CEA) level and tumor location of CRC patients affected the tumorigenesis rate of PDX model (P<0.01). The biology of CRC-PDX transplanted tumor tissue was highly consistent with that of the patient's tumor tissue. All four chemotherapy regimens could inhibit tumor growth and cause tumor tissue damage. Propofol could inhibit diarrhea in mice and protect intestinal mucosa. Conclusions The CRC-PDX model established in this study may better keep biological characteristics of primary tumors and be used as a reference model for individualized treatment of CRC patients. The malignant degree of the primary tumor is the main factor affecting the tumorigenesis rate of PDX model.
    Platycodon grandiflorum saponin D enhances the sensitivity of human rectal cancer cell strain SW1463/Oxa with drug resistance to oxaliplatin
    YAN Jun, FENG Yong'an, SHI Yongkui, ZHAO Zhihao, NI Huailiang, YANG Weizhen
    2023, 43(4):  603-607.  doi:10.16352/j.issn.1001-6325.2023.04.0603
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    Objective To investigate the enhancing effect of platycodon grandiflorum saponin D on the sensitivity of human rectal cancer drug-resistant cell line SW1463 to oxaliplatin (Oxa). Methods Human rectal cancer cell line SW1463 was collected to establish a stable oxaliplatin-resistant cell strain SW1463/Oxa. The constructed cell strain SW1463/Oxa was randomly divided into platycodon grandiflorum saponin D group, Oxa group, platycodon saponin D+Oxa group and control group. The cell proliferation curve was detected by MTT. Deoxyribonucleic acid methyltransferase 3a (DNMT3a), phosphorylated histone γ-H2AX, DNA double strand repair protein RAD51 and signal transducer and activator of transcription 3(STAT3) messenger RNA (mRNA) expression were detected by RT-qPCR. DNMT3a, γ-H2AX, RAD51, STAT3 protein expression and phosphorylated STAT3(p-STAT3) level were detected by Western blot. Results Human rectal cancer drug resistant cell strain SW1463/Oxa was successfully constructed. Compared with the control group and Oxa group, the cell proliferation, DNMT3a, RAD51, STAT3 mRNA and protein expression and p-STAT3 level of platycodon saponin D group and platycodon saponin D+Oxa group decreased(P<0.05), and the expression of γ-H2AX mRNA and protein increased(P<0.05). Conclusions Platycodon grandiflorum saponin D may enhance the chemo-sensitivity of human rectal cancer drug-resistant cell strain SW1463/Oxa to Oxa, and the potential mechanism is inhibition of expression of DNMT3a, RAD51, STAT3 and promotion of expression of γ-H2AX.
    Decitabine combined with arsenic trioxide inhibits proliferation and promotes apoptosis of acute myeloid leukemia cell lines
    LIU Yue, CAO Yang, GU Weiying, SHANG Limei, LIU Yan
    2023, 43(4):  608-614.  doi:10.16352/j.issn.1001-6325.2023.04.0608
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    Objective To investigate the effects of decitabine (DAC) combined with arsenic trioxide (ATO) on proliferation and apoptosis of AML cells and its underlying molecular mechanism. Methods AML cell lines were treated with DAC and ATO alone or in combination. CCK8 was used to detect cell viability;Compusyn software was used to analyze the synergistic effects of the combination treatment;Flow cytometry was applied to determine the apoptosis and cell cycle distribution;Western blot was applied to detect the expressions of poly (ADP-ribose) polymerase (PARP), c-PARP, phosphatidylinositol 3-kinase (PI3K), p-PI3K, protein kinase B (Akt) and p-Akt. Results DAC and ATO alone inhibited AML cell proliferation in a concentration-dependent manner. The combination of the two drugs significantly inhibited cell proliferation, induced apoptosis and cycle arrest of AML cells (P<0.05). Combination therapy reduced the phosphorylation level of PI3K and Akt (P<0.05). Conclusions DAC combined with ATO may have anti-AML effect by inhibiting PI3K/Akt pathway, which provides new theoretical basis of DAC+ATO strategy for AML.
    Effect of miR-140-5p targeting HMGB1/IκB-α axis on retinopathy of diabetic rats
    ZHANG Mengyao, NIU Shu, CAI Jing, ZHAO Zhigang, ZHOU Yan
    2023, 43(4):  615-620.  doi:10.16352/j.issn.1001-6325.2023.04.0615
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    Objective To investigate the effect of miR-140-5p on retinopathy in diabetic rats through targeted regulation of high mobility group protein 1(HMGB1)/nuclear factor κB inhibitory protein-α(IκB-α) axis. Methods Diabetic rats were randomly divided into model group, miR-140-5p agomir group, agomir-NC group, and control group with 10 in each. Serum and retinal tissue were isolated, and the expression of miR-140-5p and HMGB1 mRNA in serum and retinal tissue was detected by RT-qPCR. HE staining was used to evaluate pathological changes. The level of serum monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-ɑ(TNF-ɑ) and intercellular adhesion molecule-1(ICAM-1) was detected by ELISA. Retinal cell apoptosis was examined by TUNEL staining. Dual luciferase reporter gene assay was used to detect the targeting relationship between miR-140-5p and HMGB1. The protein expressions of HMGB1, IκB-α and NF-κb p65 in retinal tissue were detected by Western blot. Results HMGB1 was the target gene of miR-140-5p. The content of MCP-1, TNF-ɑ, and ICAM-1, the expression of HMGB1 and NF-κB p65 , apoptotic cells counting and tissue damage in the model group and agomir-NC group were found to be all higher than those in the control group. The miR-140-5p agomir group was lower than that in the model group(P<0.05). The expression of miR-140-5p and IκB-α in the model group and agomir-NC group were lower than those in the control group and the miR-140-5p agomir group was higher than that in the model group(P<0.05). Conclusions Up-regulation of miR-140-5p may negatively regulate HMGB1 and improve the expression of IκB-α to alleviate retinopathy of diabetic rats.
    EBV lytic proteins BZLF1 and BILF1 inhibit the expression of major histocompatibility complex in Raji cells
    ZHENG Hongling, GUO Qingwei, ZHANG Wei, ZHANG Yan, HE Xingbo, YE Bing
    2023, 43(4):  621-625.  doi:10.16352/j.issn.1001-6325.2023.04.0621
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    Objective To investigate whether BZLF1 and BILF1 can inhibit the expression of major histocompatibility complex(MHC)molecules in Raji cells during Epstein-Barr virus (EBV) lytic cycle. Methods Reactivation was triggered by treating Raji cells with TPA and NaB. As the marker protein during EBV lytic state, the BZLF1 protein expression was detected by Western blot. The expression of BZLF1 and BILF1 at transcription level was analyzed by RT-qPCR in EBV reactivation. The expression of MHC class Ⅱ and MHC class Ⅰ molecules was analyzed by flow cytometry. After BZLF1 and BILF1 over-expression or gene silencing via siRNA in Raji cells by electroporation, the expression of MHC class Ⅱ and MHC class Ⅰ molecules was detected by flow cytometry and Western blot respectively. Results EBV reactivation can be triggered with TPA and NaB. The BZLF1 and BILF1 gene expression was observed by inducing EBV reactivation in Raji cells. The levels of MHC class Ⅱ and MHC class Ⅰ molecules were down regulated by inducing EBV reactivation or ectopic expression of BZLF1 and BILF1 (P<0.05). Expression of MHC class Ⅱ and MHC class Ⅰ molecules was restored by specific siRNA. Conclusions BZLF1 and BILF1 down regulate the expression of MHC class Ⅱ and MHC class Ⅰ molecules in EBV reactivation respectively.
    Effect of heat shock transcription factor 5(Hsf5) knockdown on heat shock family in mouse Leydig cells and Sertoli cells
    ZHONG Lin, ZHONG Hongli, DAI Yujie, ZHANG Qinghua
    2023, 43(4):  626-631.  doi:10.16352/j.issn.1001-6325.2023.04.0626
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    Objective To investigate the effect of knockdown heat shock transcription factor 5(Hsf5) on the expression of heat shock family in mouse Leydig cells (TM3) and Sertoli cells(TM4). Methods Mouse TM3 and TM4 cells were cultured and randomly divided into control group and Hsf5 knockdown group. The expression of heat shock transcription factor(HSF) and heat shock protein(HSP) mRNA in the two groups of cells was detected by real-time quantitative PCR. According to the results of RT-qPCR, HSPs with significant differences in the two kinds of cells were selected (P<0.01). Western blot were used to detect the expression of HSPA2, HSPA5, HSP90ab1 in TM3 cells and HSPA2,HSP90aa1 in TM4 cells. Results Hsf5 knockdown model was successfully constructed by RNA interference. After knockdown of Hsf5, the mRNA expression of Hspa2,Hspa5,Hsp90ab1 in TM3 cells and Hsf2,Hspa2, Hsp90aa1, Hsp90ab1, Hspd1 in TM4 cells was all significantly down-regulated(P<0.05). In Hsf5 knockdown group, the expression of HSPA2,HSPA5 proteins in TM3 cells and HSPA2 protein in TM4 cells were significantly down-regulated (P<0.05). Conclusions HSF5 is potentially involved in spermatogenesis by regulating the expression of HSPA2 and HSPA5 in mouse Leydig cells and Sertoli cells.
    Clinical Sciences
    Sex hormone replacement therapy for patients with Prader-Willi syndrome
    WANG Liang, WANG Xi, NIE Min, WU Xueyan, MAO Jiangfeng
    2023, 43(4):  632-635.  doi:10.16352/j.issn.1001-6325.2023.04.0632
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    Objective To analyze the sex hormonal therapy present status in patients with Prader-Willi syndrome (PWS) and to provide guidance for the future sex hormone application. Methods Pubertal development, abnormal behavior, bone mineral density, sex hormone treatment and parents' attitude to sex hormone application were analyzed by a self-designed questionnaire. Results A total of 41 PWS patients were included (21 males and 20 females) and the median age was 15 years old (13-24 years). For boys, all had cryptorchidism, of which 6 patients (28.6%) had no pubertal development. None of the patients were receiving additional androgen therapy. For girls, 13 (65%) had primary amenorrhea. Only 2 patients had received or was continuing estrogen replacement therapy. Considering the inconveniences of drug application and other reasons, 16 cases (39%) of parents were unwilling to undergo sex hormone therapy. Conclusions Almost all patients with PWS are hypogonadism. Lack of medical recommendations and limited knowledge on sex hormones for their parents, may contribute to low prevalence of sex hormone therapy in this population.
    Influence of high altitude on bone age development of children and adolescents
    Cidanwangjiu, Tudan'awang, YANG Meijie, Puqiongqiongda, WANG Fengdan, PAN Hui, JIN Zhengyu
    2023, 43(4):  636-640.  doi:10.16352/j.issn.1001-6325.2023.04.0636
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    Objective To explore the influence of altitude on the bone growth and development of children and adolescents. Methods From September 2013 to December 2021, children and adolescents with normal development were included from the following regions with three different altitudes:Beijing (Peking Union Medical College Hospital) at 43.5 m above sea level, Lhasa (People's Hospital of Tibet Autonomous Region) at 3 650 m, and Nagqu (People's Hospital of Nagqu and People's Hospital of Nima County) over 4 500 m. Their demographic information and left wrist radiographs were retrospectively analyzed. Assisted by an artificial intelligence bone age (BA) software, a radiologist with 10 years of BA experience interpreted the BA using the Greulich-Pyle method. The differences between BA and chronological age (CA) in different age groups among different altitudes were calculated and statistically analyzed. Results A total of 1 013 participants (671 boys and 342 girls) consisting of 407 Han from Beijing, 456 Tibetan from Lhasa and 150 Tibetan from Nagqu were included. Generally, the BA of children and adolescents in Beijing conformed to the CA, while the average BA of Tibetan children in Lhasa and Naqu lagged behind the CA by 0.50 and 1.33 years, respectively. Compared with those in Beijing, the BA of most age groups (7-18 years old) of Tibetan boys and girls lagged further behind the CA with the increase of altitude. Conclusions The BA of Tibetan children in plateau area lags behind their CA and the gap becomes larger with the increase of altitude.
    Effect of subclinical hypothyroidism on endocrine and metabolic characteristics of patients with polycystic ovary syndrome
    ZHANG Yuanmou, HAN Kang, XIA Rui, SUN Wenjian, REN Xiaoying, WANG Yan
    2023, 43(4):  641-646.  doi:10.16352/j.issn.1001-6325.2023.04.0641
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    Objective To analyze the effect of subclinical hypothyroidism(SCH)on endocrine and metabolic characteristics of polycystic ovary syndrome(PCOS)patients, and to explore the correlation between thyroid stimulating hormone (TSH) level and various endocrine and metabolic indexes. Methods Totally 661 patients with PCOS in the outpatient Department of Endocrinology and Metabolism of the First People's Hospital of Yunnan Province from January 2020 to June 2021 were selected as the research object. The general data and related endocrine and metabolic indexes of the patients were collected. Taking the TSH value of 4.2 mIU/L as the cut-off point for the diagnosis of SCH, PCOS patients were divided into SCH group and non SCH group. The differences of endocrine and metabolic characteristics between the two groups were compared; The correlation between TSH level and its endocrine and metabolic indexes was analyzed. Results The incidence of SCH in PCOS patients was 31.16%. Compared with PCOS patients without SCH, the levels of TSH, prolactin and triglyceride in the group with SCH were significantly higher, and the levels of free thyroxin and estradiol were significantly lower (P<0.05). TSH level was negatively correlated with free thyroxin in PCOS patients (r=-0.152, P<0.001), and positively correlated with total cholesterol and triglycerides (r=0.133, 0.111, P<0.01,P<0.05, respectively). Conclusions PCOS patients with SCH have more serious endocrine hormone disorders and dyslipidemia, which may further aggravate the risk of cardiovascular diseases and reproductive abnormalities in PCOS patients. It is suggested that patients with PCOS should be screened for thyroid function and lipid metabolism regularly and followed up for a long time.
    A single center survey of anxiety and depression status in Chinese cystic fibrosis patients
    ZHOU Wangji, SUN Yanyan, CHENG Chongsheng, ZHAO Yang, HAN Jiangna, DAI Jinrong, MENG Shuzhen, XU Kaifeng, TIAN Xinlun
    2023, 43(4):  647-650.  doi:10.16352/j.issn.1001-6325.2023.04.0647
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    Objective To study anxiety and depression in Chinese patients with cystic fibrosis (CF). Methods Clinical data from 28 CF patients in the cystic fibrosis registry study of Peking Union Medical College Hospital were collected and analyzed. Hospital anxiety and depression scale was used to identify anxiety and depression. Results The cohort had 42.9% (12/28) males with a mean age of 23±4.8 years. 14 patients (50%) had anxiety or depression. Among them, 8 subjects (28.6%) had moderate and severe anxiety or depression, and 5 patients (17.9%) were with moderate and severe anxiety, and 7 patients(25%) with moderate and severe depression. One patient(3.6%) had severe anxiety, no patients with severe depression, and four patients (14.3%) had both moderate and severe anxiety and depression. Compared to patients without anxiety or depression, patients with anxiety had significantly lower FVC% pred (54.5%±18.8% with anxiety, 80.0%±14.6% without anxiety or depression, P<0.05), and patients with depression had significantly lower FEV1% pred (41.6%±21.6% with depression, 64.2%±20.9% without anxiety or depression, P<0.05) and FVC% pred (53.9%±21.0% with depression, 80.0%±14.6% without anxiety or depression, P<0.01). Conclusions Half of CF patients have anxiety or depression, with a high comorbidity rate of anxiety and depression and a low prevalence of severe anxiety or depression in our cohort. CF patients with anxiety or depression have worse pulmonary ventilationy function compared to CF patients without anxiety or depression.
    Construction of prediction model for chronic postsurgical pain after video-assisted thoracoscopic surgery
    ZHANG Le, YUAN Yuchen, ZHANG Yuelun, SHEN Le
    2023, 43(4):  651-655.  doi:10.16352/j.issn.1001-6325.2023.04.0651
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    Objective To construct a prognostic prediction model for chronic postsurgical pain after video-assisted thoracoscopic surgery and develop a risk evaluation tool. Methods As a single-center prospective study, the study enrolled 1 132 patients received video-assisted thoracoscopic surgery between November 2020 and May 2021 at Peking Union Medical College Hospital. Numeric rating scale was used to assess the severity of acute pain when they were discharged and chronic pain at 6 months after surgery. Patients were divided into two groups based on the presence or absence of chronic postsurgical pain. Multivariate Logistic regression was conducted to analyze the risk factors which were used to construct prediction model and draw nomogram for chronic postsurgical pain after video-assisted thoracoscopic surgery. Results 460 patients (40.6%) were reported chronic pain at 6 months after surgery. More women and more patients with coronary heart disease belonged to the group of chronic postsurgical pain. Patients reported higher scores of numeric rating scale when they were discharged and inclined to keep taking painkiller after discharge. However, there were fewer patients with incisions fewer than 3 or using nitrous oxide during surgery in the group of chronic postsurgical pain (P<0.05). The outcome of multivariate Logistic regression showed that the history of coronary heart disease, not using nitrous oxide in surgery, incisions more than or equal to 3, and the score of numeric rating scale on discharge were independent risk factors for chronic postsurgical pain (P<0.05). The area under the receiver operator characteristic curve of the prediction model was 0.734. Conclusions The prediction model is conducive to clinical management for chronic postsurgical pain after video-assisted thoracoscopic surgery.
    Case Report
    Thyroid storm caused by subacute thyroiditis after Cushing's syndrome remission:one case report
    YU Jie, HUANG Qibin, LI Yuxiu, LI Mei, LIAN Xiaolan, PING Fan
    2023, 43(4):  656-659.  doi:10.16352/j.issn.1001-6325.2023.04.0656
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    A 46-year-old female was subjected to unilateral left adrenalectomy due to Cushing's syndrome 5 months ago. After surgery, she was placed on glucocorticoid substitution and discontinued the treatment due to nausea and vomiting 3 weeks ago. Then she developed subacute thyroiditis and progressed to thyroid storm. Her condition worsened after administration of propylthiouracil and β-blockers, but was soon relieved by administration of glucocorticoids.
    Mini Reviews
    Research progress on the role of kallikrein 8 in neurological diseases
    LIU Shiyu, LIU Yujian
    2023, 43(4):  660-664.  doi:10.16352/j.issn.1001-6325.2023.04.0660
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    Kallikrein 8(KLK8) is closely related to the central nervous system and is involved in the pathological processes of many neurological diseases by altering inter-synaptic adhesion relationships and extracellular matrix molecules thereby modifying synaptic morphology and regulating synaptic plasticity. In this paper, we review the role and pathogenesis of KLK8 in neurological disorders such as dysthymic disorders, Alzheimer's disease, epilepsy, and multiple sclerosis with respect to its physiological properties. It is expected to provide a theoretical basis for the diagnosis and prognosis of neurological diseases and provide directions for the research of such diseases.
    Advances of researches on astrocytes in inflammatory diseases of central nervous system
    LI Liya, QIN Pei
    2023, 43(4):  665-668.  doi:10.16352/j.issn.1001-6325.2023.04.0665
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    Astrocytes respond to various forms of central nervous system (CNS) damage which are activated to be reactive astrocytes and participate in tissue repairing, initiation and progression of neuroinflammatory diseases in CNS through neuroprotective or neurotoxic effects. The mechanism of reactive astrocytes is mainly related to their phenotypic transformation and activation of downstream inflammatory pathways. Exploring the characterization of reactive astrocytes and the underling mechanisms may provide unique insights into the pathogenesis of neuroinflammatory diseases and to identify potential targets of clinical treatment.
    Research progress on inflammatory mechanism of diabetic neuropathy
    JIANG Ye, YU Zhuoying, LI Min
    2023, 43(4):  669-673.  doi:10.16352/j.issn.1001-6325.2023.04.0669
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    Diabetic neuropathy is a common complication of diabetes, but the mechanism remains unclear. Recent studies have shown that inflammation may play a critical role in the development and progression of diabetic neuropathy. In the chronic low-grade inflammatory state of diabetes, the inflammatory signaling pathways in neurons and their surrounding cells are activated and interact with the released inflammatory factors to promote the occurrence of diabetic neuropathy. Advances in the understanding of inflammatory mechanism will facilitate the discovery of the potential therapeutic targets for the development of diabetic neuropathy.
    Progress in gene therapy mechanism of amyotrophic lateral sclerosis
    LI Xiaoguang, YANG Lu, LIU Xudong, JIA Xinmiao, YANG Xinzhuang, CUI Liying
    2023, 43(4):  674-679.  doi:10.16352/j.issn.1001-6325.2023.04.0674
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    Gene therapy research on amyotrophic lateral sclerosis (ALS) focuses on SOD1, C9ORF72, FUS and ATAX2, et al. According to the mechanism, the pathogenicity of variant genes is mostly acquired by the toxicity gain of function. Two strategies are adopted for the silencing and editing of disease causing genes, namely, naked injection of antisense oligonucleotides (ASO) to induce the degradation of mRNA mediated by RNase H or the silencing of disease causing gene mediated by adeno-associated virus (AAV) to delivery RNA interference (RNAi) to decrease toxicity protein. CRISPR/Cas9 edits and modifies the causing gene to decrease the expression of toxic protein. In addition, there has been much progress in decreasing the level of toxic protein through specific antibodies and metabolic regulators, as well as neuroprotective therapy.
    Progress in the exosomes mediating SARS-CoV-2 infection and transmission
    CUI Shiyu, ZHANG Haifeng
    2023, 43(4):  680-684.  doi:10.16352/j.issn.1001-6325.2023.04.0680
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    Exosomes play vital roles in the pathogenesis of COVID-19 infection. The infection and transmission of the disease is regulated by exosomes and their components, such as nucleic acids, proteins and other substances. The exosomes with COVID-19 particles are produced via the endosomal pathway from the infected cells, and mediate the viral infection and transmission to healthy cells, finally ending with the results of tissue damage and multiple organ dysfunctions. As the key mediators of intercellular communication, exosomes activate the immune system and enhance the response against virus invasion. In addition, COVID-19 may escape from immunity with the help of exosomes; this may be a potential way of reinfection. The role of exosomes in novel coronavirus infection and transmission provides important clues for the development of potential technology for the diagnosis and prevention.
    Research progress on the mechanism of perivascular adipose tissue inflammation promoting atherosclerosis
    DU Fenghe, LIU Bao
    2023, 43(4):  685-689.  doi:10.16352/j.issn.1001-6325.2023.04.0685
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    Atherosclerosis is harmful to human health. Under pathological conditions including hypertension and hyperlipidemia, increased levels of pro-inflammatory immunocytes in perivascular adipose tissue (PVAT), “whitening” of brown adipose tissue, and oxidative stress could promote the development of PVAT inflammation. PVAT inflammation could induce various crucial stages of atherosclerosis by disturbing secretion of inflammatory and anti-inflammatory adipokines, inhibiting PVAT cell autophagy, and activating angiogenesis in arterial adventitia, thereby promoting the onset and progression of atherosclerosis.
    Research progress on the role of HMGB1 signal pathway in bronchial asthma
    SUN Ying, YANG Zhian, HE Yao, YU Qin
    2023, 43(4):  690-694.  doi:10.16352/j.issn.1001-6325.2023.04.0690
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    High mobility group box 1 protein (HMGB1), as a ubiquitous nuclear protein, expresses in almost all cells. After activation and release, HMGB1 binds to its receptor and mediates pulmonary inflammatory response. HMGB1 signaling pathway is closely related to the occurrence and development of bronchial asthma. Related targeted drug therapy has been carried out in animal test and in clinical trials.
    Research progress on the role of adipokines in the pathogenesis of sarcopenia in the elderly
    DANG Caihong, LI Weixin
    2023, 43(4):  695-699.  doi:10.16352/j.issn.1001-6325.2023.04.0695
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    The change of fat distribution, the decrease of physical activity and the influence of diseases during aging will change the circulating level of adipokines. Adipokines may promote inflammation or anti-inflammation response, and keep glucose homeostasis and energy metabolism by regulating inflammatory reaction. Adipokines activate or inhibit the signal transduction pathway of substance metabolism, and play a role in the occurrence and development of muscle attenuation of aging organs. Paying attention to the role of adipokines in muscle attenuation will support the prevention and treatment of sarcopenia in the elderly.
    Advance in susceptibility genes and epigenetic regulation in Hirschsprung's disease
    WANG Taiyao, ZHENG Lifei
    2023, 43(4):  700-705.  doi:10.16352/j.issn.1001-6325.2023.04.0700
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    Hirschsprung's disease(HSCR)is a disease resulted from abnormal enteric nervous system development. The abnormal coding of related genes may affect the migration, proliferation, differentiation or survival of neural crest cells in digestive tract and then result in distal intestinal aganglionosis. The regulation of neural crest cells and surrounding environment involves various genes, signal pathways, transcription factors and epigenetic mechanisms. Therefore, the mutation of related genes and the change of gene expression during the development of intestinal nervous system may be related to the pathogenesis of Hirschsprung's disease. This article reviews the relevant mechanisms involved in the development of enteric nervous system, and summarizes the main genes and epigenetic patterns related to Hirschsprung's disease, such as RET, EDNRB and DNA methylation, which can affect the development of neural crest disease. The review also summarizes the main pathogenesis of Hirschsprung's disease, providing a theoretical basis for the study of Hirschsprung's disease and recommends a new strategy for the prevention and treatment of Hirschsprung's disease.
    Research progress on the role of NLRP3 inflammasome in the treatment of ischemia-reperfusion injury
    LU Yapeng, XIE Jianqin
    2023, 43(4):  706-710.  doi:10.16352/j.issn.1001-6325.2023.04.0706
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    NOD-like receptor protein 3(NLRP3) inflammasome, as a protein complex that constitutes the innate immune system, can mediate inflammatory responses by regulating the maturation and release of IL-1β and IL-18, which are the basic for multiple pathological injury. A variety of injury factors generated during ischemia-reperfusion injury (I/RI) can activate the NLRP3 inflammasome, resulting in the excessive release of IL-1β and IL-18, which in turn leads to an inflammatory cascade to aggravate the damage of various tissues and organs. Inhibition of NLRP3 inflammasome activation has become an effective method to alleviate organ I/RI. Therefore, exploring the detailed mechanism of NLRP3 inflammasome-mediated I/RI can provide a theoretical basis for the prevention and treatment of organ I/RI.