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Apelin alleviates the damage of renal podocytes induced by high glucose

Liyan WANG ; Fei AN ; Zongli DIAO ; Hongdong HUANG ; Wenhu LIU

Basic & Clinical Medicine.2023;43(12):1771-1777. doi:10.16352/j.issn.1001-6325.2023.12.1771

Objective To explore the protective effect of polypeptide Apelin on podocyte mitochondria in diabetic nephropathy and underling mechanisms.Methods Human renal podocytes were divided into four experimental groups:control group,high glucose(HG)group(glucose 25 mmol/L,48 h),Apelin intervention HG group(Ape-lin-13 1 μmol/L,48 h)and Apelin group(Apelin-13 1 μmol/L,48 h).The podocyte apoptosis was observed by TUNEL staining,the expression of mitochondrial membrane protein FUNDC1 was detected by Western blot,and the binding of mitochondrial fission protein DRP1 to FUNDC1 was examined by immunoprecipitation.The 8-week-old male mice were divided into three experimental groups:control group,diabetes group(intraperito-neal injection of streptozotocin 150 mg/kg,only one time)and Apelin intervention DM group(intraperitoneal injection of Apelin-13 0.3 μmol/kg,daily).The morphology of renal was observed by PAS staining and trans-mission electron microscopy.Plasma creatinine(Cr),urea nitrogen,urinary albumin and creatinine were de-tected by ELISA kit.The level of creatinine clearance rate(Ccr)and urinary albumin/creatinine(ACR)was calculated.Results Compared with the control group,the podocyte apoptosis and expression of FUNDC1 in the HG group increased significantly(P＜0.05),and the combination of mitochondrial fission division protein DRP1 to FUNDC1 raised.Meanwhile,compared with the HG group,the number of apoptosis,the expression of FUNDC1(P＜0.05),and the combination of DRP1 to FUNDC1 all reduced in Apelin intervention HG group.Animal experiments showed that the kidney structure of the control group was intact.In the DM group,the num-ber of podocytes decreased significantly,the foot processes were fused and dropped off.In the Apelin intervention DM group,podocyte lesions were less severe than those in the DM group.Compared with the control group,the level of plasma Cr,BUN and urine ACR in the DM group increased,while the level of Ccr decreased significantly(P＜0.05).However,compared with the DM group,the level of above biomarkers in the Apelin intervention DM group was improved(P＜0.05).Conclusions Apelin keeps mitochondrial homeostasis and reduces podocyte ap-optosis by inhibiting the expression of mitochondrial membrane protein FUNDC1,which may contribute to allevia-tion of diabetic nephropathy.

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Screening for NOTCH2NLC gene dynamic mutation in patients with essential tremor

Xinyi ZHANG ; Ximeng ZHAO ; Yingmai YANG ; Xinhua WAN ; Liying CUI ; Xue ZHANG ; Qing LIU

Basic & Clinical Medicine.2023;43(12):1778-1783. doi:10.16352/j.issn.1001-6325.2023.12.1778

Objective To identify the pathogenic variants in 110 patients with essential tremor(ET).Methods Clinical data and peripheral blood samples of ET patients were collected from the Department of Neurology of Peking Union Medical College Hospital and then the genomic DNA was extracted.Dynamic mutation detection of NOTCH2NLC was performed in patients with essential tremor by triplet repeat primed PCR(TP-PCR).Since ET is as-sociated with multiple mechanisms of neuro-degeneration,the next generation sequencing(NGS)panel targeting neu-rodegenerative associating genes were performed to check pathogenic variants in additional genes.Results A total of 110 ET patients and 187 matched control individuals were recruited.The age of onset in the current ET group was(36.30±17.64)years,and 74.8%patients had a family history.No abnormal trinucleotide repeat expansion in NOTCH2NLC was identified.The repeat number of(GGC)n lied within normal ranges between 10－47(average 18.6±5.4).Variants burden analysis showed association of ET with PLA2G6.Three rare variants in four patients in PLA2G6 were identified with unknown significance.Conclusions Dynamic mutations of NOTCH2NLC are uncom-mon in ET patients and that suggests need of more researches for further exploring the genetic mechanism of ET.

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The role of GPS2 in regulating proliferation,migration and invasion of hepatocellular carcinoma cell line MHCC-97H

Aixia LIU ; Hao ZHANG ; Jie SUN ; Lihua YANG ; Xiaotao ZHAO

Basic & Clinical Medicine.2023;43(12):1784-1791. doi:10.16352/j.issn.1001-6325.2023.12.1784

Objective To explore the role of G protein pathway suppressor 2(GPS2)in regulating proliferation,migration and invasion in hepatocellular carcinoma cell line MHCC-97H.Methods The expression of GPS2 in hepatocellular carcinoma(HCC)cells were detected using quantitative PCR or Western blot;GPS2 was either over-expressed or knocked down in HCC cells using over-expression vectors or short hairpin RNAs(shRNAs)for GPS2,respectively.Clone formation assay,scratch assay and Transwell assay were used to detect the effects of GPS2 on proliferation,migration and invasion of HCC cells.Results In HCC cells,over-expression of GPS2 was found to inhibit the proliferation,migration and invasion of HCC cells MHCC-97H,while knockdown of GPS2 up-regulated proliferation,migration and invasion of MHCC-97H(P＜0.05).The over-expression of GPS2 in MHCC-97H cells was found to inhibit the expression of cell proliferation,migration and invasion-related factors such as EGFR,MMP1,MMP3 and MMP9,while knockdown of GPS2 upregulated the expression of these factors(P＜0.05).Conclusions GPS2 is a novel HCC regulatory factor that inhibits proliferation,migration and invasion of HCC cells.

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Chemokine CX3CL1 has potential anti-fibrosis effect in mouse liver fibrosis

Qi CHENG ; Ning LI ; Kangkang YU ; Guangfeng SHI

Basic & Clinical Medicine.2023;43(12):1792-1795. doi:10.16352/j.issn.1001-6325.2023.12.1792

Objective To explore the effect and mechanism of chemokine CX3CL1 on mouse liver fibrosis model.Methods C57BL/6 mice were randomly divided into CCl-4 model group and normal control group with 8 animals in each group.The model group was injected with 10%CCl-4 intra peritoneally for 6 weeks.After 6 weeks,the mice were sacrificed,and the pathological changes of the mouse liver were observed by HE staining.The level of CX3CL1 in peripheral blood of the mice was measured,and the expression level of CX3CL1 mRNA in the liver tis-sue of the mice was detected.In addition,in order to explore the mechanism of CX3CL1,the level of IFN-γ in mouse serum was detected as well.Results After the 6-week modeling,the liver pathology microscopy showed a successful modeling of liver fibrosis.The serum CX3CL1 level and liver tissue CX3CL1 expression in the model group were found to be significantly up-regulated,which suggested a potential impact on the pathogenesis of liver fi-brosis.In addition,the level of IFN-γ in the serum of mice in the model group increased significantly.Conclusions CX3CL1 is related to liver fibrosis in mice,and its mechanism might be explained by promoting the production of IFN-γ so to prevent liver fibrosis.

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Establishment of reference intervals of squamous cell carcinoma antigen for healthy population in Nanning region

Dongyi ZHOU ; Yuhong WEI ; Liling YI ; Shangmou WEI ; Chunling ZHU ; Sufang YANG ; Qiliu PENG

Basic & Clinical Medicine.2023;43(12):1796-1800. doi:10.16352/j.issn.1001-6325.2023.12.1796

Objective To establish a reference intervals(RIs)of serum squamous cell carcinoma antigen(SCC-Ag)in healthy population in Nanning region and provide clinical evidence to support diagnosis and prognosis of squamous cell carcinoma.Methods A total of 10 197 reference individuals who joined a routine physical examina-tion in the Health Management Center of Guangxi International Zhuang Medical Hospital from March 2019 to De-cember 2021 were collected.The level of serum SCC-Ag was detected by chemiluminescence microparticle immuno-assay.The Mann-Whitney U test was applied to compare the differences in serum SCC level between genders or ad-jacent age groups.The unilateral 95th percentile determined the upper limit of the RIs by the nonparametric method.Another 1 035 healthy subjects with the same conditions as the reference population were selected for refer-ence validation.Results The serum SCC-Ag level showed a skewed distribution(Z＝0.08,P＜0.05).The ser-um SCC-Ag level of males was considerably higher than that of females.There was significant difference in serum SCC-Ag level between males aged 18－30 and 31－40,51－60 and 61－90(P＜0.05).There was significant difference in serum SCC-Ag level between females aged 18－30 and 31－40,31－40 and 41－50,51－60 and 61－ 90(P＜0.05).The reference intervals of serum SCC-Ag was as follows:0－1.64 ng/mL for males and females aged 18－30 years;0－1.57 ng/mL and 0－1.70 ng/mL for males aged 31－60 years and 61－90 years,respec-tively;0－1.50 ng/mL,0－1.52 ng/mL and 0－1.42 ng/mL for females aged 31－40 years,41－60 years and 61－90 years,respectively.Conclusions The RIs of serum SCC-Ag in healthy population in the Nanning region are successfully established according to different genders and ages.

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Daidzein alleviates high glucose-induced pyroptosis of renal tubular epithelial cells

Caiwei CHEN ; Yueping WU ; Zhuansuo WANG ; Qiang LUO ; Bozhen XING

Basic & Clinical Medicine.2023;43(12):1801-1807. doi:10.16352/j.issn.1001-6325.2023.12.1801

Objective To investigate the impact of daidzein(DAI)on the pyroptosis of renal tubular epithelial cells induced by high glucose by regulating NOD-like receptor protein 3(NLRP3)/caspase-1 signal pathway.Methods HK-2 cells were divided into control group(NC group)(5.5 mmol/L D-glucose),HG group(30 mmol/L D-glucose),DAI-L,DAI-M,DAI-H groups(HK-2 cells were incubated with 30 mmol/L D-glucose and 25,50,100 μmol/L DAI,respectively),and DAI-H+LPS group(HK-2 cells were incubated with 30 mmol/L D-glucose,100 μmol/L DAI and 1 μg/mL LPS).MTT assay was applied to detect the cytotoxicity and proliferation of HK-2 cells;the apoptosis of HK-2 cells was detected by flow cytometry.The level of interleukin-1β(IL-1β)and inter-leukin-18(IL-18)in HK-2 cells was detected by ELISA.The morphology of pyroptosis cells was ob-served by scanning electron microscope.Immunofluorescence staining was applied to detect pyroptosis related pro-teins.The expression of NLRP3,cleaved casase-1 and GSDMD-N was detected by Western blot.Results In NC group,the cells were spherical with regular boundaries,while in HG group,the cells swelled and became larger with irregular boundaries;the OD value(490 nm)of HK-2 cells in HG group was obviously lower than that in NC group(P＜0.05),the apoptosis rate,IL-1β,IL-18 contents,NLRP3,cleaved casase-1,GSDMD-N protein level of HK-2 cells were obviously higher(P＜0.05);After DAI treatment,the swelling of cells was alleviated,the A value(490 nm)of HK-2 cells increased significantly(P＜0.05),the apoptosis rate of HK-2 cells,IL-1 β,IL-18 content,NLRP3,cleaved-caspase-1 and GSDMD-N protein levels were significantly decreased(P＜0.05)and the therapeutic effect of DAI was dose-dependent;LPS eliminated the beneficial effect of DAI-H on high glucose in-duced apoptosis of renal tubular epithelial cells.Conclusions DAI may alleviate pyroptosis of renal tubular epithe-lial cells induced by high glucose through inhibition of NLRP3/caspase-1 signaling pathway.

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Effects of miR-34a on polarization and inflammatory factor content of mouse macrophage cell line RAW264.7 under high glucose conditions

Fengyun ZHANG ; Zhihao ZHAO ; Zhuoqi ZHANG

Basic & Clinical Medicine.2023;43(12):1808-1813. doi:10.16352/j.issn.1001-6325.2023.12.1808

Objective To detect the role of miR-34a on macrophage inflammation and polarization under high glucose conditions.Methods Mouse macrophages were collected and cultured during high glucose for 3－28 days.RT-qPCR was used to detect the expression of miR-34a,iNOS,MCP-1 and Arg-1 mRNA.Then miR-34a was over-expressed or silenced,ELISA was used to detect the expression of IL-6,IL-1β,TNF-α and qRT-PCR was used to detect the expression of iNOS and MCP-1 mRNA.Western blot was used to detect the expres-sion of Notch1.Results Expression of miR-34a increased under high glucose conditions in RAW264.7 cells continuously.Over-expression of miR-34a promoted the expression of MCP-1 and iNOS observed by RT-qPCR and increased the expression of IL-6,IL-1β and TNF-α detected by ELISA.Further studies showed that siRNA-Notch1 down-regulated the expression of miR-34a,MCP-1,iNOS,IL-6,IL-1β and TNF-α.Conclusions Chronic high glucose condition stimulates the expression of miR-34a which promotes M1 macrophage polarization and releasing of pro-inflammatory factors.

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Over-expression of APMAP alleviates glomerular podocyte injury in adriamycin nephropathy

Jingyi WU ; Wenjing FAN ; Donghua YAN

Basic & Clinical Medicine.2023;43(12):1814-1821. doi:10.16352/j.issn.1001-6325.2023.12.1814

Objective To investigate the effects of adipocyte plasma membrane-associated protein(APMAP)over-expression on glomerular podocyte injury in adriamycin(ADR)nephropathy.Methods The rat model of adriamy-cin nephropathy was constructed by tail vein injection of adriamycin,the expression of APMAP and NF-κB p65 in renal tissue was measured by immunohistochemistry.A mouse glomerular podocytes MPC-5 cell line with APMAP gene over-expression was constructed,then podocyocytes injury model was induced by 0.5 μmol/L ADR and trea-ted with NF-κB signaling pathway activator CU-T12-9.The proliferation of cells was checked by CCK-8.The activity of lactate dehydrogenase(LDH)was determined by ELISA.The apoptosis of podocytes was determined by flow cytometry.Western blot was used to detect protein expressions of NF-κB p65,p-NF-κB p65 and TNF-α.Results APMAP was expressed in kidney tissue of doxorubicin nephropathy rats at a low level,while NF-κB p65 was significantly high expressed(P＜0.05).Over-expression of APMAP increased proliferation of MPC-5 cells and decreased LDH activity,apoptosis rate,and also down-regulated protein expression of NF-κB p65,P-NF-κB p65 and TNF-α under ADR exposure(P＜0.05).However,combined treatment with CU-T12-9 significantly inhibited the ameliorative effect of APMAP over-expression on the damage of MPC-5 cells exposed to ADR.Conclusions The over-expression of APMAP can inhibit ADR-induced glomerular podocyte injury,and its mechanism might be related to the inhibition of NF-κB signaling pathway.

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Esketamine reduces ischemia-reperfusion injury of skeletal muscle of rats

Peigen YUAN ; Shunli CHEN ; Yuanlu SHAN ; Binbin XUE ; Yuzhu YE ; Lina LIN

Basic & Clinical Medicine.2023;43(12):1822-1826. doi:10.16352/j.issn.1001-6325.2023.12.1822

Objective To investigate the role of low dose esketamine in pretreated limb ischemia-reperfusion inju-ry.Methods The rats were divided into sham-operated group(Sham group),ischemia-reperfusion group(I/R group,3 h of ischemia and 2 h of reperfusion),esketamine group(ESK group,ip,5 mg/kg).The plasma con-centrations of creatine kinase(CK)and lactate dehydrogenase(LDH)were measured.The wet/dry weight ratio of skeletal muscle was immediately detected.The gastrocnemius muscle was harvested and the level of malondial-dehyde(MDA)and superoxide dismutase(SOD)was detected by colorimetric assay.The expression of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)were detected by Western blot and im-munohistochemical staining.Results Compared with Sham group,the index of wet/dry weight ratio,MDA,CK,LDH,Nrf2 and HO-1 were all increased,but SOD was decreased in I/R group(P＜0.05).Compared with I/R group,the index of wet/dry weight ratio,MDA,CK and LDH were significantly lower,but SOD and Nrf2 and HO-1 were significantly higher in ESK group(P＜0.05).Conclusions ESK may increase Nrf2 in the nucleus,thereby increase the HO-1 protein as an antioxidant agent.

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Alpinetin alleviates bleomycin-induced pulmonary fibrosis in mice

Changxing SI ; Yanyan DING ; Fengxian YIN

Basic & Clinical Medicine.2023;43(12):1827-1833. doi:10.16352/j.issn.1001-6325.2023.12.1827

Objective To investigate the effect and potential mechanism of alpinetin(ALPN)on bleomycin(BLM)-induced pulmonary fibrosis in mice.Methods The mice were randomly divided into control group,model group(intratracheally instilled BLM),low-dose(L-ALPN group)and high-dose ALPN groups(H-ALPN group)(10 mg/kg or 30 mg/kg ALPN daily,respectively)with 10 in each.Lung tissues were collected,and the alveolar structure and pathological morphology were microscopied by HE and Masson staining;mRNA and protein expres-sions of collagen Ⅰ,TGF-β1,E-cadherin,α-SMA,p-PERK,PERK,CHOP and GRP78 in lung tissue were de-tected by RT-qPCR,immunohistochemistry and Western blot,respectively.Results Compared with control group,the lung of the model group showed fibrotic changes,and the expression of collagenⅠ,TGF-β1,α-SMA,p-PERK/PERK,CHOP and GRP78 in lung tissue was significantly increased(P＜0.01).E-cadherin expression was significantly decreased(P＜0.01).Compared with model group,pulmonary fibrosis was significantly alleviated in low and high doses ALPN groups,the expression of collagenⅠ,TGF-β1,α-SMA,p-PERK/PERK,CHOP and GRP78 in lung tissue were significantly decreased(P＜0.05 or P＜0.01),and the expressionof E-cadherin was sig-nificantly increased(P＜0.05 or P＜0.01).Conclusions ALPN may alleviate BLM-induced pulmonary fibrosis,and this effect may be attributed to the inhibition of endoplasmic reticulum stress.

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