Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

Objective:To investigate the mechanism of JAK2/STAT3/SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods:36 SPF SD rats were randomly divided into 6 groups,with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group,with density wave,frequency 2Hz-50 Hz,intensity 2 mA,muscle tremor as the degree 20 min/time,1 time/day,for 3 consecutive days.Observe the general condition of rats;the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4(IL-4)and interleukin-8(IL-8)were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2,STAT3,SOCS1 protein and mRNA in rat colon tissue.Results:In contrast to the normal group,the overall condition of the model group was worse,the colonic mucosa was severely damaged,even necrotic,and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced,and the content of IL-8 was obviously go up(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously go up,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced(P<0.01).In contrast to the model group,the general condition of rats in each acupoint group was significantly improved,the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated,the content of IL-4 in serum was obviously go up,and the content of IL-8 was significantly decreased(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously reduced,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up(P<0.05,P<0.01).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced,the content of serum IL-4 was significantly increased,and the content of IL-8 was significantly decreased(P<0.05,P<0.01).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated,while the protein content and mRNA expression of SOCS1 were significantly go up(P<0.05,P<0.01).Conclusion:Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli(ST36)is better than that of Tianshu(ST25),Dachangshu(BL25)and Shangjuxu(ST37).The mechanism may be related to the regulation of JAK2/STAT3/SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

扶正解毒方对病毒性心肌炎大鼠氧化应激及Sirt1/FoxO3a通路的影响

目的 基于沉默信息调节因子2相关酶1(Sirt1)/叉头蛋白O3a(FoxO3a)通路探究扶正解毒方对病毒性心肌炎(VMC)大鼠的影响。方法 大鼠腹腔注射柯萨奇B3病毒(CVB3)法建立VMC模型,随机分为模型组、扶正解毒方低剂量组(28 g/kg)、扶正解毒方高剂量组(112 g/kg)、EX527组(Sirt1抑制剂,1μg/kg)、扶正解毒方(28 g/kg)+EX527(1μg/kg)组,每组15只,另取未造模的正常大鼠15只为正常组,连续给药10 d。检测大鼠超声心动图Tie指数,HE染色观察心肌组织病理损伤情况,ELISA法检测血清心肌损伤标志物、氧化应激水平,TUNEL法检测心肌组织细胞凋亡率,免疫组化法检测心肌组织Sirt1表达,Western blot法检测心肌组织FoxO3a、磷酸化FoxO3a(p-FoxO3a)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)、过氧化氢酶(CAT)、细胞周期抑制蛋白p27、半胱氨酸天冬氨酸蛋白水解酶3(caspase3)表达。结果 与正常组比较,模型组大鼠心肌细胞坏死加重,大鼠出现死亡,Tie指数、血清心肌损伤标志物、氧化应激指标水平均升高(P<0.05),Sirt1/FoxO3通路蛋白及其介导的抗氧化应激、抗凋亡相关蛋白表达降低(P<0.05);与模型组比较,扶正解毒方各剂量组大鼠无死亡,心肌组织损伤得以缓解,Tie指数、血清心肌损伤标志物、氧化应激指标水平均降低(P<0.05),Sirt1/FoxO3通路蛋白及其介导的抗氧化应激、抗凋亡相关蛋白表达升高(P<0.05);EX527可加重VMC大鼠的死亡及心肌细胞氧化损伤等症状,并逆转扶正解毒方对抗病毒性心肌炎的作用(P<0.05)。结论 扶正解毒方可通过激活Sirt1/FoxO3a通路缓解VMC大鼠心肌氧化应激损伤。

川陈皮素通过FOXO3a/SIRT1通路减轻脂多糖诱导的肺损伤

目的 观察川陈皮素在脓毒症肺损伤中的作用,并探究其潜在机制。方法 采用随机数字表法将48只8~10周的雄性C57 BL/6小鼠分为4组,即对照组、肺损伤组、治疗组(5mg/kg)和治疗组(10mg/kg),每组各12只。对照组不做任何处理;肺损伤组小鼠腹腔注射脂多糖(10mg/kg);治疗组(5mg/kg)小鼠接受腹腔注射脂多糖(10mg/kg)的同时予以川陈皮素(5mg/kg)灌胃;治疗组(10mg/kg)小鼠接受腹腔注射脂多糖(10mg/kg),同时予以川陈皮素(10mg/kg)灌胃。脂多糖腹腔注射12h后,检测各组小鼠肺功能及动脉血气,同时留取肺组织,分别从病理学和分子生物学层面评估小鼠肺损伤状况及可能靶点。结果 与对照组比较,肺损伤组小鼠气道压力、PaCO_(2)、肺损伤评分明显增高,PaO_(2)明显降低(P<0.05);与肺损伤组比较,治疗组(5mg/kg)和治疗组(10mg/kg)小鼠气道压力、PaCO_(2)、肺损伤评分明显降低,PaO_(2)明显升高(P<0.05)。Western blot法检测结果显示,与对照组比较,肺损伤组小鼠肺组织中IL-1β、TNF-α、TXNIP和4-HNE的蛋白表达水平明显升高(P<0.05);与肺损伤组比较,治疗组(5mg/kg)和治疗组(10mg/kg)小鼠肺组织上述蛋白表达水平明显降低(P<0.05)。此外,肺损伤组小鼠肺组织中FOXO3a和SIRT1的蛋白表达水平较对照组明显降低(P<0.05);与肺损伤组比较,治疗组(5mg/kg)和治疗组(10mg/kg)小鼠肺组织中FOXO3a和SIRT1蛋白表达水平明显上调(P<0.05)。结论 川陈皮素可抑制小鼠脓毒症肺损伤并减轻氧化应激和炎性反应,其机制可能与川陈皮素对FOXO3a/SIRT1通路激活有关。

Mechanism of Resveratrol on autophagy mediated by Mst1/Sirt3 signaling pathway in diabetic cardiomyopathy

Objective:To observe the effects of resveratrol on myocardial cell injury and Mst1/Sirt3 signaling pathway mediated autophagy in type 2 diabetic mice. Methods:C57 BL/KSJ db/db mice were allocated to the normal control group,the model group,and the resveratrol group;C57 BL/KSJ db/m mice served as the melbine group,with 10 mice each. The resveratrol group and the melbine group were treated with resveratrol and metformin by gavage,respectively. The normal control group and the model group were treated with equal volume of normal saline by gavage,for 8 consecutive weeks. H & E staining,transmission electron microscopy and immunofluorescence were used to observe the pathological morphology,ultrastructure and apoptosis levels of myocardial tissues,respectively. RT-qPCR method was used to detect the expression levels of apoptosis genes Bax and Bcl-2 in myocardial tissues,and Western-blot method was used to detect the expression levels of autophagy proteins(LC3 and p62),Mst1 and Sirt3 proteins in myocardial tissue. Results:Compared with the model group,resveratrol can significantly reduce the body weight,blood glucose level and serum CK and LDH levels of db/db mice,and the differences were statistically significant(P<0.05;P<0.01). Meanwhile,after resveratrol treatment,myocardial inflammation score,apoptosis rate,Bax mRNA expression level and Bax/Bcl-2 ratio in myocardial tissue were significantly reduced,and Bcl-2 mRNA expression level was significantly increased,and the differences were statistically significant(P<0.01). In addition,compared with the model group,the expression level of p62 and p-Mst1 protein in the myocardial tissue of the resveratrol group was significantly reduced,and the expression level of Sirt3 protein and the ratio of LC3Ⅱ/LC3Ⅰ were significantly increased,and the differences were statistically significant(P<0.01). Conclusion:Resveratrol promotes the autophagy level of cardiomyocytes by activating the Mst1/Sirt3 signaling pathway and inhibits cardiomyocyte apoptosis to play a protective role in diabetic cardiomyopathy.

基于SIRT1/FOXO3a信号通路探讨“温阳通脉”灸法对ApoE^(-/-)动脉粥样硬化小鼠炎症反应的影响

【目的】探讨“温阳通脉”灸法防治动脉粥样硬化(AS)的作用机制。【方法】将10只饲喂普通饲料的C57BL/6J小鼠设为空白组;将30只ApoE^(-/-)小鼠给予高脂饲料喂养建立动脉粥样硬化模型,随机分为模型组、辛伐他汀组和艾灸组,每组10只。于造模第1天开始干预,艾灸组小鼠给予膻中、神阙、内关、血海穴艾灸,辛伐他汀组给予辛伐他汀蒸馏水混悬液灌胃,共干预12周。给药结束后,采用苏木素-伊红(HE)染色法观察小鼠胸主动脉病理形态结构,透射电镜观察小鼠胸主动脉内皮细胞超微结构,酶联免疫吸附法(ELISA)检测小鼠血清肿瘤坏死因子α(TNF-α)、细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)水平,实时定量聚合酶链反应(qRT-PCR)法检测胸主动脉沉默信息调节因子1(SIRT1)和叉头框转录因子O3a(FOXO3a)的mRNA表达水平,Western Blot法检测胸主动脉SIRT1和FOXO3a蛋白表达水平。【结果】与空白组比较,模型组小鼠胸主动脉及血管内皮细胞病理变化明显,血清炎症因子TNF-α、ICAM-1和VCAM-1水平升高(P<0.05或P<0.01),胸主动脉SIRT1 mRNA和蛋白表达量降低(P<0.01),FOXO3a mRNA和蛋白表达量差异无统计学意义(P>0.05);与模型组比较,辛伐他汀组和艾灸组小鼠的胸主动脉及血管内皮细胞结构得到明显改善,血清TNF-α、ICAM-1、VCAM-1水平均降低(P<0.05),胸主动脉SIRT1 mRNA和蛋白表达量增加(P<0.05或P<0.01),FOXO3a mRNA和蛋白表达量差异均无统计学意义(P>0.05)。辛伐他汀组和艾灸组上述指标组间比较,差异均无统计学意义(P>0.05)。【结论】“温阳通脉”灸法可通过调控SIRT1/FOXO3a信号通路减轻炎症反应防治小鼠AS。

Endogenous hydrogen sulfide and ERK1/2-STAT3 signaling pathway may participate in the association between homocysteine and hypertension

Background Homocysteine(Hcy)is a risk factor for hypertension,although the mechanisms are poorly understood.Methods We first explored the relationship between Hcy levels and blood pressure(BP)by analyzing the clinical data of primary hypertensive patients admitted to our hospital.Secondly,we explored a rat model to study the effect of Hcy on blood pressure and the role of H2S.An hyperhomocysteinemia(HHcy)rat model was induced to explore the effect of Hcy on blood pressure and the possible mechanism.We carried out tissue histology,extraction and examination of RNA and protein.Finally,we conducted cell experiments to determine a likely mechanism through renin-angiotensin-aldosterone system(RAAS)and extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway.Results In primary hypertensive inpatients with HHcy,blood pressure was significantly higher as compared with inpatient counterparts lacking HHcy.In the rat model,blood pressure of the Wistar rats was significantly increased with increases in serum Hcy levels and decreased after folate treatment.Angiotensin converting enzyme 1(ACE1)expression in the Wistar Hcy group was enhanced comparing to controls,but was decreased in the Wistar folate group.Angiotensin II receptor type 1(AGTR1)levels in the kidney tissue increased in the Wistar folate group.Both serum H2S and kidney cystathionineγ-lyase decreased with elevated levels of serum Hcy.In vitro,increased concentrations and treatment times for Hcy were associated with increased expression of collagen type 1 and AGTR1.This dose and time dependent response was also observed for p-STAT3 and p-ERK1/2 expression.Conclusion Endogenous H2S might mediate the process of altered blood pressure in response to changes in serum Hcy levels,in a process that is partly dependent on activated RAAS and ERK1/2-STAT3 signaling pathway.

shRNA-interfering LSD1 inhibits proliferation and invasion of gastric cancer cells via VEGF-C/PI3K/AKT signaling pathway

BACKGROUND Histone Lysine Specific Demethylase 1(LSD1)is the first histone demethylase to be discovered,which regulates various biological functions by making lysine of histone H3K4,H3K9 and non-histone substrates demethylated.Abnormal regulation of LSD1 is closely related to the occurrence and development of gastric cancer.The change of LSD1 expression level plays an important role in the proliferation and metastasis of gastric cancer cells.The study of its function and mechanism may provide a theoretical basis for early diagnosis and targeted therapy of gastric cancer.AIM To investigate the effect of downregulation of lysine-specific demethylase 1(LSD1)expression on proliferation and invasion of gastric cancer cells and the possible regulatory mechanisms of the VEGF-C/PI3K/AKT signaling pathway.METHODS The LSD1-specific short hairpin RNA(shRNA)interference plasmid was transiently transfected,and expression of LSD1 was downregulated.The cell proliferation ability of LSD1 was observed by CCK-8 assay after downregulating expression of LSD1.Transwell invasion assay was used to observe the change of cell invasion ability after downregulating expression of LSD1.Expression of phosphorylated phosphoinositide 3-kinase(p-PI3K),PI3K,p-AKT,AKT,vascular endothelial growth factor receptor(VEGFR)-3,matrix metalloproteinase(MMP)-2 and MMP-9 in each group was detected by Western blotting.RESULTS The cell proliferation ability of transiently transfected LSD1-shRNA interference plasmid group was significantly lower than that of the control group(P<0.05).Transwell invasion assay showed that the number of cells across the membrane of the LSD1-shRNA transfection group(238.451±5.216)was significantly lower than that of the control group(49.268±6.984)(P<0.01).Western blotting showed that expression level of VEGF-C,p-PI3K,PI3K,p-AKT,AKT,VEGFR-3,MMP-2 and MMP-9 in the LSD1-shRNA group was significantly lower than that in the control group(P<0.05).CONCLUSION Downregulation of LSD1 expression inhibits metastatic potential of gastric cancer cells,and VEGF-C-mediated activation of PI3K/AKT signaling pathway,which may be an important mechanism for inhibiting lymph node metastasis in gastric cancer cells.

3-epi-bufotalin suppresses the proliferation in colorectal cancer cells through the inhibition of the JAK1/STAT3 signaling pathway

Traditional Chinese medicine(TCM)has been increasingly employed in the last decades in China for both preventing and treating a variety of cancers.3-epi-bufotalin is an active ingredient of TCM“Chanpi”with anti-tumor potential.However,the effect and mechanism of 3-epi-bufotalin on colorectal cancers were not well disclosed.The present study demonstrated that 3-epi-bufotalin could reduce viability,trigger apoptosis,and block the cell cycle at the G2/M stage in colorectal cancer cell lines HT29,RKO,and COLO205 in vitro.Moreover,3-epi-bufotalin inhibited the JAK1/STAT3 signaling pathway.These results indicated the anti-proliferation ability of 3-epi-bufotalin in colorectal cancer cells.

Baicalin protects neonatal rat brains against hypoxicischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway

Baicalin is a flavonoid compound extracted from Scutellaria baicalensis root.Recent evidence indicates that baicalin is neuroprotective in models of ischemic stroke.Here,we investigate the neuroprotective effect of baicalin in a neonatal rat model of hypoxic-ischemic encephalopathy.Seven-day-old pups underwent left common carotid artery ligation followed by hypoxia（8% oxygen at 37°C） for 2 hours,before being injected with baicalin（120 mg/kg intraperitoneally） and examined 24 hours later.Baicalin effectively reduced cerebral infarct volume and neuronal loss,inhibited apoptosis,and upregulated the expression of p-Akt and glutamate transporter 1.Intracerebroventricular injection of the phosphoinositide 3-kinase/protein kinase B（PI3 K/Akt） inhibitor LY294002 30 minutes before injury blocked the effect of baicalin on p-Akt and glutamate transporter 1,and weakened the associated neuroprotective effect.Our findings provide the first evidence,to our knowledge that baicalin can protect neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the PI3 K/Akt signaling pathway.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

芪白平肺胶囊通过调节SIRT1/FoxO3a通路改善COPD气虚痰瘀证大鼠炎症及氧化应激状态

目的探讨慢性阻塞性肺疾病(COPD)气虚痰瘀证大鼠的氧化应激及炎症反应与沉默信息调节因子1/叉头框转录因子O3a(SIRT1/FoxO3a)通路的调节及芪白平肺胶囊对其影响。方法雄性SD大鼠80只,随机分组为正常组、模型组、芪白平肺胶囊组及金水宝对照组,每组20只;采用复合因素建立COPD气虚痰瘀证病证结合大鼠模型,药物处理后,使用ELISA测定各组大鼠血清中超氧化物气化酶(SOD)、丙二醛(MDA)、白细胞介素1β(IL-1β)、 IL-2的水平,荧光定量PCR检测大鼠肺组织SIRT1 mRNA及FoxO3a mRNA水平,采用Western blot法测量各组大鼠肺组织SIRT1及FoxO3a蛋白。结果与正常组比较,模型组的MDA、 IL-1β、 IL-2的水平显著升高、 SOD水平显著降低, SIRT1的mRNA和蛋白水平降低, FoxO3a的mRNA和蛋白水平升高;与金水宝对照组比较,芪白平肺胶囊组MDA、 IL-1β、 IL-2水平降低, SOD水平升高, SIRT1的mRNA和蛋白表达升高, FoxO3a的mRNA和蛋白表达降低。结论芪白平肺胶囊能够通过调节SIRT1/FoxO3a通路改善COPD气虚痰瘀证炎症及氧化应激状态。

通络醒脑泡腾片经Nampt/SIRT1/FOXO3途径改善SAMP8小鼠的学习记忆

目的考察通络醒脑泡腾片(川芎、当归和黄芩)对SAMP8小鼠学习记忆的影响,探究其改善认知功能作用机制。方法将SAMP8小鼠药物干预60 d,采用Morris水迷宫评价其认知变化,随后采用ELISA法和免疫组化法分别测定脑组织超氧化物歧化酶(SOD)、羰基化蛋白、NAD^+/NADH和海马尼克酰胺磷酸核糖转移酶(Nampt)、沉默调节蛋白1(SIRT1)和叉头蛋白3(FOXO3)的表达。结果通络醒脑泡腾片能够缩短SAMP8小鼠在隐藏平台实验中第5天的逃避潜伏期和显著增加平台象限所占时间百分比,明显增加SAMP8小鼠在空间探索实验中进入目标象限次数;能够明显提升SAMP8小鼠脑组织SOD活力和NAD^+/NADH比值及降低羰基化蛋白浓度;能够显著上调SAMP8小鼠海马Nampt和SIRT1的表达,下调FOXO3的蛋白表达。结论通络醒脑泡腾片提高SAMP8小鼠认知功能与其调节海马Nampt/SIRT1/FOXO3途径有关。

CTRP3通过激活SIRT1/FOXO3a途径减轻OGD/R诱导的心肌细胞损伤

目的:探究CTRP3在糖氧剥夺/再灌注(OGD/R)诱导的心肌细胞损伤中的作用及其可能的作用机制。方法:体外培养大鼠心肌细胞H9c2,OGD/R诱导H9c2细胞损伤。qRT-PCR法检测细胞CTRP3、SIRT1和FOXO3a mRNA表达;Western blot检测细胞CTRP3、SIRT1、FOXO3a、Bax、Bcl-2和cleaved caspase-3蛋白表达,CCK-8检测细胞存活,流式细胞术检测细胞凋亡,检测细胞CK-MB、cTnI、SOD和MDA活性、GSH-Px含量及ROS的产生。结果:与Control组相比,OGD/R组细胞中CTRP3、SIRT1和FOXO3a mRNA以及蛋白表达,细胞存活率、Bcl-2蛋白表达、SOD和GSH-Px活性明显降低,细胞凋亡率、Bax和cleaved caspase-3蛋白表达、CK-MB、cTnI和MDA含量、ROS荧光强度明显升高(P<0.05)。OGD/R组与oe-NC组细胞各指标比较,差异无统计学意义(P>0.05)。与oe-NC组相比,oe-CTRP3组细胞中CTRP3、SIRT1和FOXO3a mRNA以及蛋白表达,细胞存活率、Bcl-2蛋白表达、SOD和GSH-Px活性明显升高(P<0.05),细胞凋亡率、Bax和cleaved caspase-3蛋白表达、CKMB、cTnI和MDA含量、ROS荧光强度明显降低(P<0.05)。与oe-CTRP3组相比,oe-CTRP3+EX527组细胞中SIRT1、FOXO3a和Bcl-2蛋白表达、细胞存活率、SOD和GSH-Px活性明显降低,细胞凋亡率、Bax和cleaved caspase-3蛋白表达、CK-MB、cTnI和MDA含量、ROS荧光强度明显升高(P<0.05)。与oe-CTRP3+EX527组相比,oe-CTRP3+EX527+oe-FOXO3a组细胞中FOXO3a和Bcl-2蛋白表达,细胞存活率、SOD和GSH-Px活性明显升高,细胞凋亡率、Bax和cleaved caspase-3蛋白表达、CKMB、cTnI和MDA含量、ROS荧光强度明显降低(P<0.05)。结论:CTRP3通过激活SIRT1/FOXO3a途径促进OGD/R诱导的心肌细胞存活,抑制细胞凋亡和氧化应激。

Cetirizine regulates scleroderma skin fibrosis in mice via the TGF-β1/Smad3 signaling pathway

Objective:To investigate the effect of cetirizine on the fibrosis of skin tissue in systemic sclerosis(SSc)mice and its mechanism of action.Methods:Thirty-two BALB/C mice were randomly divided into a blank group,a model group,a cetirizine low-dose group,and a cetirizine high-dose group,with eight in each group.The blank group was injected with normal saline on the back,and the other three groups were injected with bleomycin on the back to prepare SSc mouse models.The mice were injected once a day for 28 consecutive days,while the normal group and the model group were given saline.The dose group was administrated intragastrically at 2 mg/kg and 5 mg/kg,respectively,for 28 consecutive days.Detect the thickness of the dermis by taking the skin tissue in the back injection area of each group.Hematoxylin-eosin staining(HE)and Masson staining.Sample hydrolysis method to detect hydroxyproline(HYP)content in skin tissue.Immunohistochemical detection ofα-smooth muscle actin(α-SMA)expression in skin tissues.Enzyme-linked immunosorbent assay(ELISA)to detect serum interleukin(IL-6,IL-10)and transforming growth factor(TGF-αand TGF-β1).Quantitative real-time PCR(qRT-PCR)was used to detect the expression levels of collagen type I(COL1A1),type III collagen(COL3A1),Smad homolog 3(Smad3),and TGF-β1 mRNA.Western blot was used to detect the expression levels of COL1A1,COL3A1 and p-Smad3.Results:Compared with the blank group,the dermis thickness and HYP content of the model group increased,the skin tissue lesions and fibrosis were more severe,theα-SMA positive expression intensity in the skin tissue was higher,and the serum IL-6,IL-10,TGF-α,TGF-β1 content increased,COL1A1,COL3A1,Smad3,TGF-β1 mRNA expression levels increased in skin tissues,COL1A1,COL3A1,p-Smad3 protein expression increased,the differences were statistically significant(P<0.05).Compared with the model group,the dermal thickness and HYP content of the low and high dose cetirizine groups were reduced,the degree of skin tissue lesions and fibrosis was improved,the expression ofα-SMA in skin tissues was weakened,the levels of IL-6,IL-10,TGF-α,TGF-β1 in serum were reduced,the expression levels of COL1A1,COL3A1,Smad3 and TGF-β1 in skin tissues were reduced,and the expression levels of COL1A1,COL3A1,and p-Smad3 proteins were reduced,the decrease in the high-dose group was more significant,and the differences were statistically significant(P<0.05).Conclusion:Cetirizine can improve the degree of fibrosis of skin tissue in SSc mice and reduce the immune inflammation response.The mechanism of action is related to the TGF-β1/Smad3 signaling pathway.

SIRT1/FOXO3a/p27信号通路在白藜芦醇抑制肺动脉平滑肌细胞增殖中的作用

目的探讨白藜芦醇(RES)是否通过影响沉默信息调节因子2相关酶1(SIRT1)/叉头蛋白O3a(FOXO3a)/p27信号通路抑制肺动脉平滑肌细胞(PASMC)增殖,为肺动脉高压新药研发提供一定的理论和实验依据。方法体外培养大鼠PASMC并随机分为三组,对照组不处理,5-HT刺激组加1μmol/L 5-HT,不同浓度RES干预组在加入5-HT前用10、30、100、300μmol/L RES预处理1 h,作用24 h后采用BrdU掺入法检测各组细胞增殖情况并筛选RES作用浓度;为研究机制进一步将PASMCs分为六组,对照组、5-HT刺激组、RES干预组(筛选出的RES作用浓度)处理同前,RES+SIRT1抑制剂干预组、RES+FOXO3a抑制剂干预组在加入1μmol/L 5-HT前用RES分别联合1μmol/L EX527、AS1842856预处理1 h,RES+p27 siRNA转染组在加入1μmol/L 5-HT前预先转染p27 siRNA 24 h后联合RES;作用24 h后采用BrdU掺入法检测各组细胞增殖情况,采用Western blotting法检测对照组、5-HT刺激组、RES干预组、RES+SIRT1抑制剂干预组的FOXO3a、p27蛋白及RES+FOXO3a抑制剂干预组的p27蛋白。结果PASMC增殖率5-HT刺激组高于对照组,100、300μmol/L RES干预组均低于5-HT刺激组(P均<0.05),故以100μmol/L作为后续实验中RES干预的作用浓度。PASMC增殖率5-HT刺激组高于对照组,RES干预组低于5-HT刺激组;RES+SIRT1抑制剂干预组、RES+FOXO3a抑制剂干预组、RES+p27 siRNA转染组均高于RES干预组(P均<0.05),三组间差异无统计学意义。PASMC中FOXO3a、p27蛋白表达量5-HT刺激组均低于对照组,RES干预组均高于5-HT刺激组,RES+SIRT1抑制剂干预组均低于RES干预组(P均<0.05);RES+FOXO3a抑制剂干预组p27蛋白表达量低于RES干预组(P<0.05)。结论RES可通过激活SIRT1上调FOXO3a,进而上调细胞周期抑制蛋白p27,最终抑制PASMC增殖。

Mechanism of hesperidin improving myocardial ischemia/reperfusion injury in type 2 diabetic rats through SIRT1/Nrf2/HO-1 signaling pathway

Objective:To observe the protective effect of hesperidin on myocardial ischemia/reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods:50 Sprague-Dawley(SD)rats were randomly assigned to the normal control group(NC),model group,ischemia-reperfusion group(IR),hesperidin group,SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group.In addition to NC,the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats.After then,the myocardial ischemia/reperfusion injury(MIRI)rat model was established by LAd for 30 minutes with 2 hours reperfusion.He staining was used to observe the pathological changes of myocardial tissue,and the levels of serum LDH,CK-MB and SOD,GSH and MDA in myocardial tissue were detected by kit methods,and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot;Results:Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration,reduce LDH activity,CK-MB and MDA level,and increase SOD activity,GSH and 4-HNE level,the differences were statistically significant when compared with IR group(P<0.01).In addition,compared with the ischemia-reperfusion group,the expressions of SIRT1,Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated,the differences were statistically significant(P<0.01);Conclusion:Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway,and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

Simiao Wan alleviates obesity-associated insulin resistance via PKCε/IRS-1/PI3K/Akt signaling pathway based on network pharmacology analysis and experimental validation

Background:The purpose of the study was to investigatethe active ingredients and potential biochemicalmechanisms of Simiao Wan(SMW)in obesity-associated insulin resistance.Methods:An integrated network pharmacology method to screen the active compoundsand candidate targets,construct the protein-protein-interaction network,and ingredients-targets-pathways network was constructed for topological analysis to identify core targets and main ingredients.To find the possible signaling pathways,enrichment analysis was performed.Further,a model of insulin resistance in HL-7702 cells was established to verify the impact of SMW and the regulatory processes.Results:An overall of 63 active components and 151 candidate targets were obtained,in which flavonoids were the main ingredients.Enrichment analysis indicated that the PI3K-Akt signaling pathway was the potential pathway regulated by SMW in obesity-associated insulin resistance treatment.The result showed that SMW could significantly ameliorate insulin sensitivity,increase glucose synthesis and glucose utilization and reduce intracellular lipids accumulation in hepatocytes.Also,SMW inhibited diacylglycerols accumulation-induced PKCεactivity and decreased its translocation to the membrane.Conclusion:SMW ameliorated obesity-associated insulin resistance through PKCε/IRS-1/PI3K/Akt signaling axis in hepatocytes,providing a new strategy for metabolic disease treatment.

Effect and Mechanism of Dicliptera chinensis Polysaccharide on miR-141/AMPK/SIRT1 Signaling Pathway in Rats with NAFLD

[Objectives]Non-alcoholic fatty liver disease(NAFLD)rat model was established by feeding high-fat and high-sugar fodder to rats,and the protective effect of Dicliptera chinensis polysaccharide(DCP)on NAFLD rats was studied to explore its potential mechanism.[Methods]45 SD rats were randomly divided into 4 groups:normal control group,model control group and DCP treatment groups(100 and 300 mg/kg).The rats in the normal control group were fed with ordinary fodder,and the rats in other groups were fed with high-fat and high-sugar diet for 14 weeks to establish NAFLD model.From the 9^(th)week,the rats in the DCP treatment groups were given different doses of DCP by intragastric administration(5 mL/kg)for 6 weeks.After the last intragastric administration,the rats fasted for 16 h,and the serum and liver of rats were collected for detection.Hematoxylin-eosin(HE)staining was conducted to observe the histopathological changes of rat liver,and alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor(TNF-α)and micrornA-141(micro RNA-141)were detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of SIRT1 and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)in rat liver was detected by western blot.[Results]Compared with the model control group,the inflammatory damage and steatodegeneration of rats in the DCP groups were relieved to varying degrees,and the number of lipid vacuoles significantly reduced.The ALT,AST,TC,TG and LDL-C content in the serum and MDA content in the liver tissue decreased to varying degrees,while the HDL-C,SOD and GSH-Px content increased.The expression of SIRT1 and AMPK increased,while the expression of miR-141,TNF-α,IL-6 and IL-1βdeclined,and the DCP 300 mg/kg treatment group had better improvement effect.[Conclusions]DCP had a certain protective effect on NAFLD rats,which may be related to the regulation of miR-141/AMPK/SIRT1 signaling pathway.

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.