Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.

抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用

目的探讨敲低长链非编码RNA(lncRNA)牛磺酸上调基因1(TUG1)抑制核苷酸结合寡聚结构域样受体蛋白1(NLRP1)炎症小体在缓解阿尔茨海默病进展中的作用。方法选取9~10周龄遗传背景为C57/BL6的野生型小鼠(WT组,10只)或淀粉样前体蛋白(APP)/早老素1(PS1)转基因小鼠(30只)。APP/PS1转基因小鼠随机分为模型(model)组,模型+敲低lncRNA TUG1组[model+lncRNA TUG1短发夹RNA(shRNA)组]和model+shRNA非靶标(NT)组,每组10只。分别采集12周龄第1天(3月龄)和32周龄第1天(8月龄)小鼠外周血和脑皮质组织,并分离皮质中的原代小胶质细胞和原代星形胶质细胞,每个时间点每组5只小鼠。Real-time PCR分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和巨噬细胞移动抑制因子(MIF)mRNA的水平,以及原代星形胶质细胞中补体蛋白C1r和C1s mRNA的水平。ELISA法测定其外周血浆中MIF含量。对3月龄和8月龄小鼠脑皮质原代小胶质细胞和原代星形胶质细胞共培养。CCK-8法测定上述2种细胞的增殖能力。Western blotting分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织中MIF、白细胞介素1β前体(pro-IL-1β)、凋亡相关斑点样蛋白(ASC)、Caspase-1(p20)、Caspase-1(full)、NLRP1及NLRP3蛋白的表达水平。采用免疫荧光染色法测定8月龄各分组小鼠脑皮质组织中β淀粉样蛋白(Aβ)表达。结果3月龄和8月龄时,与WT组小鼠相比,model组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF相对表达水平显著上调,原代小胶质细胞和原代星形胶质细胞增殖能力增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF的相对表达水平显著降低,原代小胶质细胞和原代星形胶质细胞增殖能力降低(P<0.05)。与WT组相比,model组小鼠外周血浆中MIF含量显著升高;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)、NLRP1以及NLRP3的蛋白表达水平显著升高;Aβ免疫荧光强度明显增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠外周血浆中MIF含量显著降低;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)和NLRP1的蛋白表达水平显著降低,Aβ免疫荧光强度明显降低(P<0.05),而NLRP3蛋白质的表达水平无明显变化(P>0.05)。与model组相比,model+shRNA NT组小鼠上述所有检测指标差异均无显著性(P>0.05)。结论APP/PS1转基因小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF因子表达上调与脑皮质内NLRP1炎症小体激活成正相关,敲低lncRNA TUG1可缓解阿尔茨海默病的进展。

基于线粒体动力学途径探究小分子Sigma-1受体对顺铂诱导的急性肾损伤模型的治疗作用

目的:基于线粒体动力学途径变化下探究小分子Sigma-1受体对顺铂(CDDP)诱导的急性肾损伤(AKI)的影响。方法:实验分为对照组、PRE-084组、CDDP组、CDDP+PRE-084组,共4组,每组10只小鼠,CDDP组和CDDP+PRE-084组经腹腔注射CDDP(15 mg/kg)诱导AKI模型,随后PRE-084组和CDDP+PRE-084组经腹腔注射PRE-084(0.6 mg/kg),每日1次,连续7 d;结束后,全自动生化仪进行血清尿素氮(BUN)与血清肌酐(Scr)水平检测,酶联免疫吸附法(ELISA)测定尿液中肾损伤分子1(KIM-1)含量,苏木精-伊红(HE)染色观察肾组织病理形态学变化,TdT介导的dUTP缺口末端标记(TUNEL)法检测肾脏细胞凋亡情况,透射电子显微镜观察肾脏组织线粒体超微结构,生物发光技术检测肾脏组织三磷酸腺苷(ATP)产生水平,免疫荧光双染测定肾脏组织中Sigma-1受体与Mfn2定位表达,蛋白质免疫印迹法(Western blot)测定肾脏组织内线粒体分裂蛋白Drp1、Fis1与线粒体融合蛋白Opa1、Mfn1的表达。结果:与CDDP组比较,CDDP+PRE-084组小鼠血清BUN和Scr水平、尿液KIM-1水平均降低(P<0.05),肾脏组织病理损伤情况得到明显改善,TUNEL阳性细胞率减少(P<0.05),线粒体肿胀、碎裂及空泡变性程度减小,形态恢复,肾脏组织ATP含量升高(P<0.05),Sigma-1受体与Mfn2荧光染色增强,Drp1、Fis1蛋白相对表达量下调且Opa1、Mfn1蛋白相对表达量上调(P<0.05)。结论:线粒体分裂与融合途径介导了CDDP所致小鼠AKI的发生,Sigma-1受体通过调控线粒体动力学途径对CDDP诱导的AKI小鼠起到保护作用。

微小RNA-103a-3p通过肿瘤蛋白53调控凋亡抑制剂1/P53对骨质疏松症的影响

目的探讨微小RNA(miR)-103a-3p调控细胞肿瘤蛋白53调控凋亡抑制剂1(TRIAP1)对成骨细胞分化以及去卵巢小鼠骨量的影响。方法MC3T3-E1细胞分为正常对照(NC)组、miR-103a-3p-NC组、miR-103a-3p模拟(mimc)组、miR-103a-3p mimic+TRIAP1-NC组、miR-103a-3p mimic+TRIAP1 mimic组。Real-time PCR检测细胞miR-103a-3p、TRIAP1、P53的mRNA表达,MTT法和流式细胞术检测细胞增殖及凋亡,免疫荧光染色和茜红素染色检测细胞骨架F-actin表达和矿化情况,ELISA检测细胞碱性磷酸酶(ALP)活性。24只雌性小鼠设为sham组、骨质疏松症(OP)组、miR-103a-3p antagonist-NC组和miR-103a-3p antagonist组,每组6只摘取双侧卵巢制备OP模型,sham组仅分离卵巢组织周围脂肪。测定骨组织miR-103a-3p、TRIAP1、P53、ALP、骨钙素(OCN)、骨桥蛋白(OPN)的mRNA表达,microCT测定骨密度(BMD)、骨矿物质含量(BMC),HE染色观察骨组织病理改变。结果细胞转染miR-103a-3p mimic后,miR-103a-3p及P53表达升高、TRIAP1表达降低,细胞增殖降低、凋亡增加,F-actin表达减弱,钙结节数量减少,ALP活性降低(P<0.01);而在增加转染TRIAP1 mimic后,以上miR-103a-3p mimics导致的结果均得到显著逆转(P<0.01)。OP组小鼠骨组织miR-103a-3p、P53表达升高,TRIAP1、ALP、OCN、OPN基因表达降低,BMD、BMC降低,骨组织结构破坏(P<0.05);miR-103a-3p antagonist组小鼠骨组织miR-103a-3p及P53表达降低,TRIAP1、ALP、OCN、OPN基因表达升高,BMD、BMC升高,骨组织结构改善(P<0.05)。结论MiR-103a-3p可介导TRIAP1/P53抑制成骨细胞增殖及矿化,而miR-103a-3p拮抗治疗可减少OP小鼠骨量丢失。

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

AIM: To characterize high-mobility group protein 1-toll-like receptor 4(HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion(I/R) injury.METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups(n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88(My D88), and anti-translocatingchain-associating membrane protein(TRIF) antibody groups. Vehicle with the control Ig G antibody, antiHMGB1, anti-My D88, or anti-TRIF antibodies(all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor(NF)-κB p65, interleukin(IL)-6, and tumor necrosis factor(TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. Inaddition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of m RNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance.RESULTS: Blocking HMGB 1, MyD 8 8, and TRIF expression by injecting anti-HMGB1, anti-My D88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81(P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38(P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63(P < 0.05) for the sham, control, anti-HMGB1, anti-My D88, and anti-TRIF groups, respectively(all in pg/m L).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of antiHMGB1, anti-My D88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

黄芩苷对慢性萎缩性胃炎小鼠JAK1、STAT3表达的影响

【目的】通过网络药理学和动物实验探讨黄芩苷对慢性萎缩性胃炎小鼠胃黏膜的修复机制。【方法】(1)应用网络药理学预测分析黄芩苷治疗慢性萎缩性胃炎的潜在关键靶点。(2)动物实验:将40只C57BL/6N小鼠随机分为正常组、模型组、维酶素组、黄芩苷组,每组10只。除正常组,其他3组小鼠采用N-甲基-N’-硝基-N-亚硝基胍(MNNG)灌胃结合饥饱失常法构建慢性萎缩性胃炎模型。给药结束后,采用苏木素-伊红(HE)染色法观察胃黏膜组织病理变化,采用酶联免疫吸附法(ELISA)检测血清中胃泌素(GAS)和前列腺素E2(PGE2)水平变化,采用实时荧光定量聚合酶链反应(qRT-PCR)法和蛋白免疫印迹(Western Blot)法检测胃黏膜组织中Janus酪氨酸激酶1(JAK1)、信号转导和转录激活子3(STAT3)的mRNA与蛋白表达水平。【结果】网络药理学结果显示,黄芩苷与核心靶点JAK1、STAT3可自发结合。动物实验结果显示:与正常组比较,模型组小鼠胃黏膜组织发生萎缩,腺体排列紊乱,存在大量淋巴细胞,胃黏膜细胞凋亡指数显著升高(P<0.05),血清中GAS与PGE2水平显著降低(P<0.05),胃黏膜组织中JAK1、STAT3的mRNA与蛋白表达水平显著升高(P<0.05);与模型组比较,维酶素组与黄芩苷组小鼠胃黏膜病变减轻,腺体排列相对整齐,结构较完整,胃黏膜细胞凋亡指数显著降低(P<0.05),血清中GAS与PGE2水平显著升高(P<0.05),胃黏膜组织中JAK1、STAT3的mRNA与蛋白表达水平显著降低(P<0.05);黄芩苷组上述各指标与维酶素组比较,差异均无统计学意义(P>0.05)。【结论】黄芩苷可有效修复慢性萎缩性胃炎小鼠胃黏膜病变,其机制可能与下调JAK1、STAT3的mRNA及蛋白表达有关。

双酚A通过上调Apoa 1基因的表达抑制TM3细胞睾酮合成

旨在从脂质代谢的角度探讨载脂蛋白A1(apolipoprotein A1,Apoa 1)是否介导了双酚A(bisphenol A,BPA)暴露所致小鼠睾丸间质细胞株(TM3)睾酮合成的降低。将TM3细胞随机分为不同浓度的BPA暴露剂量(0、5、10、20、40、60、80μmol·L^(-1))组,0μmol·L^(-1)BPA为对照组(CON)。给予相应剂量处理24 h后,运用CCK-8法检测TM3细胞活力,确定BPA最适染毒剂量;通过ELISA检测TM3细胞培养上清液睾酮(testosterone,T)含量;利用RT-qPCR检测TM3细胞脂质代谢相关基因Apoa1、Apoa 2(apolipoprotein A2)、Apoc 3(apolipoprotein C3)的mRNA表达水平;运用Western blot和免疫荧光方法检测APOA1蛋白表达水平;采用油红O染色观察细胞内脂滴累积情况。结果表明,20μmol·L^(-1)BPA处理24 h对TM3细胞活力无显著影响,40μmol·L^(-1)BPA处理24 h后,TM3细胞活力受到极显著抑制(P<0.01);此外,20μmol·L^(-1)BPA处理TM3细胞24 h后,培养上清液中睾酮含量极显著低于对照组(P<0.01),Apoa 1基因的mRNA表达水平及蛋白表达量极显著升高(P<0.001),但Apoa 2和Apoc 3基因的mRNA表达水平无显著变化;与对照组相比,20μmol·L^(-1)BPA处理24 h,TM3细胞的脂滴累积量极显著降低(P<0.0001)。综上,BPA可通过上调Apoa 1基因的表达水平,增强胆固醇逆向转运(reverse cholesterol transport,RCT),引起TM3细胞内的脂滴含量减少,导致TM3细胞的睾酮合成分泌降低。

The remedial effect of soluble interleukin-1 receptor type Ⅱ on endometriosis in the nude mouse model

Objective： Recent studies have shown that the local expression of soluble interleukin （IL） -1 receptor type Ⅱ （slL-1 R Ⅱ ） in endometrial tissue of women with endometriosis is decreased, and the depression of IL-1 R Ⅱ was more significant in infertile women than that in fertile women with endometriosis. In this research, we investigated the remedial effect of slL-1-R Ⅱ administration on endometriosis in the nude mouse model. Methods： Nineteen nude model mice with endometriosis were randomly divided into three groups： group A was treated by intraperitoneal administration with only slL-1 R Ⅱ for two weeks, group B was similarly treated with only IL- 1, and group C （control） was administered saline. After 2 weeks, the size of the ectopic endometrial lesions was calculated, and the expression of vascular endothelial growth factor （VEGF） and B-cell lymphoma leukemia-2 （Bcl- 2） were detected by immunohistochemistry. The IL-8 and VEGF levels in the peritoneal fluid （PF） and serum were also measured by enzyme-linked immunosorbent assay （ELISA）. Results： The mean size of ectopic endometrial lesion did not differ between the three groups （P 〉 0.05）. Compared with the control, the expression of VEGF and Bcl-2 was significantly lower in group A, and higher in group B. In the three groups, the levels of IL-8 in the PF and serum were highest in group A, and lowest in group B. Conclusion： slL-1 R Ⅱ may suppresse hyperplasia of ectopic endometriosis, perhaps by reducing the expression of certain cytokines, such as VEGF, IL-8, and Bcl-2, which could provide a new clinical strategy for the treatment of endometriosis.

Expression of Angiopoietin-1/-2 in the Process of Mouse Embryo Implantation

This study examined the expression and distribution of angiopoietin-1/-2 （Ang-1/-2） in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohistochemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups： control group, D2 group （2 days after pregnancy）, D4 group （4 days after pregnancy）, D6 group （6 days after pregnancy） and D8 group （8 days after pregnancy）, each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups （P〈0.01）. Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy （day 2）, and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice （P〈0.01）. In situ hybridization showed Ang-I mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 （P〈0.01）. It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.

Cardiotrophin-1 promotes cardiomyocyte differentiation from mouse induced pluripotent stem cells via JAK2/STAT3/Pim-1 signaling pathway

Background The induced pluripotent stem cell （iPSC） has shown great potential in cellular therapy of myocardial infarction （MI）, while its application is hampered by the low efficiency of cardiomyocyte differentiation. The present study was designed to investigate the effects of cardiotrophin-1 （CT-1） on cardiomyocyte differentiation from mouse induced pluripotent stem cells （miPSCs） and the underlying mechanisms involved. Methods The optimal treatment condition for cardiomyocyte differentiation from miPSCs was established with ideal concentration （10 ng/mL） and duration （from day 3 to day 14） of CT-1 administration. Up-regulated expression of cardiac specific genes that accounted for embryonic cardiogenesis was observed by quantitative RT-PCR. Elevated amount of a-myosin heavy chain （ct-MHC） and cardiac troponin I （cTn I） positive cells were detected by immunofluorescence staining and flow cytometry analysis in CT- 1 group. Results Transmission electron microscopic analysis revealed that cells treated with CT- 1 showed better organized sacromeric structure and more mitochondria, which are morphological characteristic of matured cardiomyocytes. Western blot demonstrated that CT-1 promotes cardiomyocyte differentiation from miPSCs partly via JAK2/STAT3/Pim-1 pathway as compared with control group. Conclusions These findings suggested that CT-1 could enhance the cardiomyocyte differentiation as well as the maturation of mouse induced pluripotent stem cell derived cardiomyocytes by regulating JAK2/STAT3/Pim-1 signaling pathway.

UVA1 irradiation inhibits fibroblast proliferation and alleviates pathological changes of scleroderma in a mouse model

The purpose of the present study was to compare the effects of different doses of ultraviolet radiation A1 （UVA1） on human fibroblast proliferation and collagen level in a mouse model of scleroderma, so as to identify appropriate irradiation doses for clinical treatment of scleroderma. Monolayer from human fibroblasts was cultured in vitro, and a mouse model of scleroderma was established by subcutaneous injection of 100 μL of 400 μg/mL bleomycin into the back of BALB/c mice for 4 weeks. The mouse models and human fibroblasts were divided into UVA1- exposed （100, 60 and 20 J/cm2） and UVA-unexposed groups. At 0, 24 and 48 h after exposure, cell proliferation and levels of hydroxyproline and collagen were detected. UVA1 irradiation was performed 3 times weekly for 10 weeks, and the pathological changes of skin tissues, skin thickness and collagen level were observed after phototherapy. Cell proliferation and the levels of hydroxyproline and collagen were inhibited after phototherapy, and there was a significant difference between the UVAl-exposed cells and UVAl-unexposed cells （P 〈 0.001）. In addition, UVA1 phototherapy improved dermal sclerosis and softened the skin, and there were significant differences between the high-dose UVA1 group and the model group, and the negative group （P 〈 0.05）. It is concluded that UVA1 radiation can reduce cell proliferation, and decrease hydroxyproline and collagen levels in a dose-dependent manner in vitro. High-dose UVA1 phototherapy has marked therapeutic effect on scleroderma in the mouse model. Decreased collagen level may be related to the reduced number and activity of cells, as well as inhibition of collagen synthesis.

HSF1/AMPK信号通路在铁死亡参与糖尿病心肌病发病的机制研究

目的:探讨热休克因子1(heat shock factor 1,HSF1)/5′-单磷酸腺苷活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)信号通路调控铁死亡对糖尿病心肌病(diabetic cardiomyopathy,DCM)发病的影响。方法:本研究为实验研究,采用空白对照与多组实验对照。H9c2细胞随机分为4组:低葡萄糖组(control,Con)、高葡萄糖组(high glucose,HG)、HG+HSF1组、HG+HSF1+化合物C(compound C,CC)组。分别对细胞进行罗丹明胶质蛋白染色、细胞线粒体(reactive oxygen species,ROS)检测和细胞脂质ROS检测,并通过Western blot分析AMPK信号表达。雄性C57/BL6小鼠随机分为4组:NC组、NC+HSF1组、DM组和DM+HSF1组,每组12只。通过超声心动图评估了小鼠心血管功能参数。结果:与Con组相比,HG组HSF1、pAMPK/AMPK水平明显下调(P=0.005、0.002),和相对细胞表面积、线粒体Fe2+水平、线粒体ROS水平、细胞脂质ROS水平明显增加(P=0.001、0.003、0.006、0.002)。与HG组相比,HG+HSF1组明显逆转了这些变化(P=0.001、0.001、0.002、0.006、0.007、0.003),但加入CC时HSF1的逆转作用明显减弱(P<0.05)。与NC组相比,DM组EF%、FS%、E/A、E′/A′和心脏组织中HSF1、pAMPK/AMPK表达明显降低(均P<0.01),和心脏组织中Fe2+、ROS、丙二醛(Malondialdehyde,MDA)水平和4-羟基壬烯酸(4-Hydroxynonenal,4-HNE)蛋白水平明显增加(P=0.004、0.003、0.001、0.004),DM+HSF1组明显逆转了这些变化。结论:HSF1在DCM病理过程中发挥心脏保护作用,其抗铁死亡作用可能与AMPK依赖性的脂质代谢和线粒体稳态调节有关。

α1-抗胰蛋白酶对未成熟脑白质损伤小鼠运动功能的影响

目的探讨α1-抗胰蛋白酶(α1-antitrypsin,AAT)对未成熟脑白质损伤小鼠成年期运动功能的影响。方法将5日龄C57BL/6J幼鼠随机分为假手术组(n=27)、缺氧缺血(hypoxia-ischemia,HI)+生理盐水组(n=27)、HI+AAT组(n=27)。通过HI法建立未成熟脑白质损伤小鼠模型。HI+AAT组分别于HI前24 h、HI后立即及HI后72 h腹腔注射AAT(50 mg/kg);HI+生理盐水组在相同时间腹腔注射相同剂量生理盐水。造模后7 d和55 d进行头颅磁共振T2加权成像扫描。2月龄时利用Catwalk步态分析系统评估成年期小鼠的静态、动态和协调性参数。结果与假手术组小鼠相比,HI损伤小鼠造模后7 d头颅磁共振T2加权像呈现高信号,可见脑白质明显损伤;造模后55 d脑白质损伤仍存在。与假手术组小鼠相比,HI+生理盐水组小鼠爪印面积、最大接触面积、平均压强、最大压强、爪印宽度、平均速度、身体速度、步幅长度、摆动速度、步态模式AA占比、爪印耦合(左后爪→左前爪)占比降低(P<0.05);HI+生理盐水组爪间距离、步态模式AB占比、位相滞后(左前爪→左后爪)占比升高(P<0.05)。与HI+生理盐水组小鼠相比,HI+AAT组小鼠平均速度、身体速度、步幅长度、摆动速度(右前爪)升高(P<0.05)。结论未成熟脑白质损伤小鼠在成年期可表现出明显运动功能障碍,而应用AAT可改善其部分运动功能。

肝细胞DEP结构域蛋白5/哺乳动物雷帕霉素靶蛋白复合物1信号轴在非酒精性脂肪肝形成中的作用

目的建立肝细胞Dishevelled/Egl-10/pleckstrin(DEP)结构域蛋白5(DEPDC5)基因(Depdc5)肝细胞特异性敲除小鼠高脂喂养模型,探讨DEPDC5/哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)信号轴对非酒精性脂肪肝的调控。方法构建肝细胞特异性敲除Depdc5^(flox/flox)模型;Alb-Cre小鼠(LKO),Depdc5^(flox/flox)小鼠(Loxp)作为对照。32只2~3月龄雄性小鼠随机分为高脂LKO组、高脂Loxp对照组、高脂+雷帕霉素LKO组及高脂+雷帕霉素Loxp对照组,每组8只。检测肝脏血清生物化学指标、脂质含量、蛋白、mRNA及病理切片,采用GraphPad Prism 8软件进行统计学分析。结果高脂喂养导致LoxP小鼠肝脏脂肪变性,LKO小鼠肝脏脂肪变性减轻但合并出现肝损伤;雷帕霉素抑制了Depdc5敲除引起的mTORC1通路激活,显著改善Loxp小鼠肝脏脂肪变性,并改善LKO小鼠的肝损伤。结论Depdc5基因敲除能够保护高脂喂养小鼠肝脏脂肪变性,雷帕霉素可以改善DEPDC5缺失诱发的肝损伤。

Mdivi-1通过修复黑质网状部线粒体改善肝性脑病小鼠运动功能

目的:探讨急性肝性脑病(AHE)模型小鼠中黑质网状部(SNr)的线粒体变化,以及线粒体分裂抑制剂Mdivi-1对AHE小鼠运动功能和SNr线粒体功能的影响。方法:使用硫代乙酰胺(TAA)腹腔注射构建小鼠的AHE模型并通过腹腔注射给予Mdivi-1处理,利用生化检测试剂盒检测小鼠血清天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)和血氨的变化,并进行旷场实验、转棒疲劳实验以及高架十字迷宫实验观察AHE小鼠的运动功能;电镜下观察SNr的线粒体结构变化,商品化试剂盒检测SNr的线粒体膜电位(MMP)以及细胞的活性氧(ROS)和ATP水平。结果:与对照组相比,AHE小鼠血清中的AST、ALT和血氨含量均增加;小鼠在旷场中的总运动距离减少,转棒疲劳实验以及高架十字迷宫实验中的运动时间均缩短;SNr的线粒体变小变圆、线粒体分裂增加,MMP降低,细胞ROS增加,ATP产生减少。使用Mdivi-1干预后,AHE小鼠血清中的AST、ALT和血氨含量均降低;小鼠在旷场中的总运动距离增加,转棒疲劳实验以及高架十字迷宫实验的运动时间均增多,SNr的线粒体变大、圆率降低,线粒体分裂减少,MMP增加,细胞ROS降低,ATP产生增多。结论:Mdivi-1可以通过修复AHE小鼠SNr的线粒体从而改善运动功能。

TGF-beta1 Transgenic Mouse Model of Thoracic Irradiation: Modulation of MMP-2 and MMP-9 in the Lung Tissue

To investigate the effects of TGF-β1 on the two gelatinases （MMP-2 and MMP-9）, and their roles in lung remodeling after irradiation-induced lung injury. Expressions of TGF-β1 were measured with western blot, and expressions of MMP-2 and MMP-9 were analyzed with zymography in a TGF-β1 transgenic mouse model after thoracic irradiation with 12 Gy. We found expressions of TGF-β1 in the lung from the transgenic mice were three folds as compared to those from control mice. With densitometrical analysis, we found a significant decrease in MMP-9 activity in lung homogenates from the transgenic mice as compared with those from non-transgenic control mice 8 weeks after sham-irradiation （relative MMP-9 activity： C： 1. 000±0. 1091; TG： 0. 4772±0. 470 （n=8, P〈0.05）. But MMP-2 was constitutively expressed in the lung homogenates from the transgenic mice as compared to those from control mice 8 weeks after sham-irradiation （relative MMP-2 activity 8 weeks after sham-irradiation： C： 1. 000±0. 1556, TG： 1. 0075±0. 1472）. Eight weeks after thoracic irradiation with 12 Gy, we observed a significant increase of MMP-2 and MMP-9 activity in lung homogenates from both transgenic and normal mice. In TGF-β1 transgenic mice relative MMP-9 activity was increased to 1. 5321±0. 2217 folds 8 weeks after thoracic irradiation with 12 Gy as compared to those after sham-irradiation （1. 000±0. 2153）, and relative MMP-2 activity was increased to 1. 7142 ± 0. 4231 folds. Our results show that TGF-β1 itself down-regulates activity of MMP-9, thereby decreases ECM degradation in lungs of TGF-β1 transgenic mice. Also we find that ionizing irradiation upregulates both MMP-2 and MMP-9 activity. Over-expressions of MMP-9 and MMP-2 after lung irradiation are involved in the inflammatory response associated with radiation-induced lung injury, and maybe further in radiation-induced lung fibrosis.

过表达神经调节蛋白1的人羊膜间充质干细胞促进小鼠皮肤创面愈合

背景:神经调节蛋白1具有促进细胞增殖、分化以及血管生长等特性。人羊膜间充质干细胞是组织工程领域重要的种子细胞,已被证实参与组织修复及再生过程。目的:构建过表达神经调节蛋白1的人羊膜间充质干细胞,探究其增殖、迁移能力以及对创面愈合的影响。方法:(1)体外分离培养人羊膜间充质干细胞并对其进行鉴定;(2)构建神经调节蛋白1过表达慢病毒,将人羊膜间充质干细胞分为空载组、神经调节蛋白1组、对照组,分别转染空载慢病毒、过表达神经调节蛋白1慢病毒,对照组不进行转染;(3)EdU实验检测各组细胞增殖能力,Transwell实验检测各组细胞迁移能力;(4)构建C57BL/6小鼠创面损伤模型,随机分成对照组、空载组和神经调节蛋白1组,每组8只,分别在创面局部多点均匀注射1 mL转染空载慢病毒或转染过表达神经调节蛋白1慢病毒的人羊膜间充质干细胞,对照组注射等量的生理盐水;(5)造模后1,7,14 d观察创面愈合情况,苏木精-伊红染色观察创面愈合组织学变化,免疫组化观察创面CD31的表达。结果与结论:(1)成功构建过表达神经调节蛋白1的人羊膜间充质干细胞,细胞内神经调节蛋白1的mRNA、蛋白表达较空载组明显上调(P<0.05);(2)过表达神经调节蛋白1促进了人羊膜间充质干细胞的迁移(P<0.01)和增殖(P<0.05);(3)过表达神经调节蛋白1的人羊膜间充质干细胞促进了小鼠创面愈合(P<0.05)和创面的血管生成(P<0.05)。结果表明,过表达神经调节蛋白1提高了人羊膜间充质干细胞的增殖和迁移能力,以及增强了促进创面愈合和创面血管生成的能力。

黄芪多糖调控Wnt1信号对糖尿病大鼠糖脂代谢紊乱影响机制研究

目的探讨黄芪多糖(astragalus polysaccharide,APS)改善糖尿病(diabetes mellitus,DM)大鼠糖脂代谢的分子机制,以及其对无翅型小鼠乳房肿瘤病毒整合位点1(Wnt1)信号的调控机制。方法采用高脂饲料诱导构建大鼠DM模型,随机分为模型组(DM)、黄芪多糖低剂量组(APS-L)、黄芪多糖高剂量组(APS-H)、激动剂组(SB216763),每组10只,另取10只正常大鼠作为对照组(Control)。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记试剂盒(TUNEL)检测大鼠肝脏和胰腺组织中的细胞凋亡,酶联吸附反应试剂盒检测大鼠血清超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的水平,5,5',6,6'-四氯-1,1',3,3'-四乙基苯丙咪唑羰花青碘化物(JC-1)染色检测胰腺组织中的线粒体膜电位,免疫荧光检测胰腺组织中葡萄糖转运子4(glucose transporter 4,GLUT4)和糖原合酶激酶3β(glycogen synthase kinase-3β,GSK-3β)的表达,油红O检测肝组织中的脂质沉积,免疫组化检测肝组织中脂肪酸合成酶(fatty acid synthase,FAS)和脂蛋白脂酶(lipoprotein lipase,LPL)的表达,Western blot实验检测大鼠肝组织中Wnt1、β-连环蛋白(β-catenin)、淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl2-associated x protein,Bax)的表达。结果与Control组相比,DM组、APS-L组、APS-H组、SB216763组动物的空腹血糖(fasting blood glucose,FBG)、总胆固醇(total cholesterol,TC)和甘油三酯(triglyceride,TG)的含量,血清中MDA的水平,肝和胰腺组织中的细胞凋亡率,肝组织中脂质沉积,肝组织中Wnt1、β-catenin、Bax的表达均明显升高;胰腺组织中的线粒体膜电位,胰腺组织中GLUT4、GSK-3β的表达,肝组织中FAS、LPL、Bcl-2的表达均明显下降。与DM组大鼠相比,APS-L组、APS-H组、SB216763组大鼠FBG、TC、TG的含量,血清中MDA的水平,肝和胰腺组织中的细胞凋亡率,肝组织中脂质沉积,肝组织中Wnt1、β-catenin、Bax的表达明显下降;胰腺组织中的线粒体膜电位,胰腺组织中GLUT4、GSK-3β的表达,肝组织中FAS、LPL、Bcl-2的表达均明显升高。与APS-L组相比,APS-H组大鼠的FBG、TC、TG的含量,血清中MDA的水平,肝和胰腺组织中的细胞凋亡率,肝组织中脂质沉积,肝组织中Wnt1、β-catenin、Bax的表达均明显下降;胰腺组织中的线粒体膜电位,胰腺组织中GLUT4、GSK-3β的表达,肝组织中FAS、LPL及Bcl-2的表达明显升高。与APS-H组相比,SB216763组动物的FBG、TC、TG的含量,血清中MDA的水平,肝和胰腺组织中的细胞凋亡率,肝组织中脂质沉积,肝组织中Wnt1、β-catenin、Bax的表达均明显升高;胰腺组织中的线粒体膜电位,胰腺组织中GLUT4、GSK-3β的表达,肝组织中FAS、LPL、Bcl-2的表达均明显下降,差异均具有统计学意义(均P<0.05)。结论黄芪多糖能明显抑制DM大鼠的氧化应激,升高胰腺组织的线粒体膜电位,降低肝组织中的脂质沉积,抑制肝和胰腺组织中的细胞凋亡率,降低动物的血糖,这可能与抑制Wnt1信号的转导有关。

Characterization of Spindlin1 isoform2 in mouse testis

Aim： To investigate the expression of Spindlin 1 （Spin First, reverse-transcription polymerase chain reaction present in mouse testis. Then the expression patterns 1） isoform2 and assess its function in mouse testis. Methods： （RT-PCR） was used to determine whether Spinl isoform2 is of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages （days 0, 7, 14, 21, 28 and 60） was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence. Results： The RT-PCR results show that Spinl isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spinl isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm. Conclusion： Spinl isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.

Role of intestinal glucagon-like peptide-1 in hypoglycemia response impairment in type 1 diabetes

This study critically examines the novel findings presented by Jin et al,which explores the role of intestinal glucagon-like peptide-1(GLP-1)in impaired counterregulatory responses to hypoglycemia in mice with type 1 diabetes.The study identifies intestinal GLP-1 as a significant determinant in the physiological responses to hypoglycemia,offering new insights into its potential implications for diabetes management.The editorial synthesizes these findings,discusses their relevance in the context of current diabetes research,and outlines potential avenues for future investigation of intestinal GLP-1 as a therapeutic target.This analysis underscores the need for continued research into the complex mechanisms underlying impaired hypoglycemia responses and highlights the potential of targeting intestinal GLP-1 pathways in therapeutic strategies for type 1 diabetes.