CHANGES IN NEUROPEPTIDES AFTER MUSIC EXPOSURE 429Cardioprotective effect of ivabradine via the AMPK/SIRT1/PGC-1αsignaling pathway in myocardial ischemia/reperfusion injuryinduced in H9c2 cell

Post-resuscitation myocardial dysfunction(PRMD)is the most severe myocardial ischemia-reperfusion injury(MIRI)and is characterized by difficult treatment and poor prognosis.Research has shown the protective effects of the rational use of ivabradine(IVA)against PRMD,however,the molecular mechanisms of IVA remain unknown.In this study,an ischemia-reperfusion injury(IRI)model was established using hypoxic chambers.The results demonstrated that pretreatment with IVA reduced IRI-induced cytotoxicity and apoptosis.IVA attenuated mitochondrial damage,eliminated excess reactive oxygen species(ROS),suppressed IRI-induced ATP and NAD+,and increased the AMP/ATP ratio.We further found that IVA increased the mRNA levels of sirtuin 1(SIRT1)and peroxisome proliferator-activated receptor-γcoactivator 1α(PGC-1α)and upregulated the expression levels of phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK,SIRT1,and PGC-1αproteins.Interestingly,no change in AMPK mRNA levels was observed.Cardiomyocyte energy metabolism significantly changed after IRI.The aim of this study was to demonstrate the cardioprotective effect of Ivabradine via the AMPK/SIRT1/PGC-1αsignaling pathway in myocardial ischemia/reperfusion injury-induced in H9c2 cell.

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

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.

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.

Yiqi Yangyin and Huatan Quyu granule can improve skeletal muscle energy metabolism in a type 2 diabetic rat model by promoting the AMPK/SIRT/PGC-1α signalling pathway

Objective:To investigate how Yiqi Yangyin and Huatan Quyu granule (YYHO) improves skeletal muscle insulin resistance in a type 2 diabetic rat model and to discover whether the molecular mechanism is related to the promotion of the AMPK/SIRT/PGC-1α signalling pathway.Methods:Rats were randomly divided into 4 groups:the normal group,the model group,the YYHQ granule group,and the pioglitazone group.The type 2 diabetic rat model was established by feeding a high-fat diet for 5 weeks along with a single intraperitoneal injection of 30 mg/kg streptozotocin (STZ).After modelling successfully,the appropriate drug was intragastrically administered to diabetic rats for 2 weeks,once per day.The YYHQ granule group was given a dose of 4.8 g/kg body weight per day,the pioglitazone group was given a dose of 1.35 mg/kg body weight per day.The doses for both groups were equivalent to the clinical equivalent dose based on a previous study.Other groups were gavaged with the same amount of saline water.Body weight,food intake,water intake,urine volume and grip strength were recorded weekly.The fasting blood glucose(FBG) was determined weekly using blood glucose test strips.The related glucose and lipid metabolism indexes,e.g.,fasting insulin (Fins),glycated haemoglobin (GHb),HOMA-IR,ISI,triglycerides (TG),total cholesterol (TC),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol (LDL-C) and free fatty acid (FFA),were determined using biochemical method.The mRNA expression levels of adenosine monophosphate-activated protein kinase (AMPK),peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α),carnitine palmitoyl transterase-1 (CPT-1),Sirtuin 1 (SIRT1),and Sirtuin 3 (SIRT3) were assessed using quantitative real-time PCR (qRT-PCR).The protein expression levels of creatine kinase (CK),Ca2+ ATPase,α-Actin,AMPK,PGC-1α and CPT-1 were determined using enzyme-linked immunosorbent assay method (ELISA).Results:Body weight decreased significantly (P <.01),food intake,water intake and urine volume increased significantly (P <.01),and grip strength decreased significantly (P <.01) in the model group compared with the normal group.The levels of FBG,Fins,GHb and HOMA-IR increased significantly (P <.01),and the ISI decreased significantly (P <.01) in the model group.The levels of TG,TC,LDL-C and FFA increased significantly (P <.05 or P <.01),and the level of HDL-C decreased significantly (P <.05) in the model group.These changes were reversed after treatment with YYHQ granule or pioglitazone.Compared with the model group,the YYHQ granule and pioglitazone groups significantly improve body weight,water intake and urine volume (P <.05 or P <.01),however,both treatments had no significant effect on food intake (P >.05).The levels of FBG,Fins,GHb,HOMA-IR and ISI were improved significantly (P <.01) and the levels of TG,TC and LDL-C were improved significantly (P <.05 or P <.01),however,both treatments had no significant effect on the levels of HDL-C and FFA (P >.05).Further results indicated that YYHQ granule significantly decreased the mRNA expression of AMPK,PGC-1α,CPT-1,SIRT1 and SIRT3 in skeletal muscle (P <.01) and the pioglitazone group showed similar effects;moreover,the protein expression levels of CK,Ca2+ATPase,α-Actin,AMPK,PGC-1α and CPT-1 in skeletal muscle significantly decreased (P <.01),however,pioglitazone had no significant effect on CK and α-Actin (P >.05).Conclusion:The possible molecular mechanism of YYHQ granule improving skeletal muscle insulin resistance in a type 2 diabetic rat model may be related to the stimulation of energy metabolism in skeletal muscle via the AMPK/SIRT/PGC-1α signalling pathway.

芒柄花苷经AMPK/SIRT1/FoxO1通路对2型糖尿病肾病大鼠肾损伤的改善作用

目的:探讨芒柄花苷经AMPK/SIRT1/FoxO1通路对2型糖尿病肾病(DN)大鼠肾损伤的改善作用。方法:用腹腔注射链脲佐菌素准备大鼠DN模型,芒柄花苷低剂量组和芒柄花苷高剂量组大鼠分别灌胃给予芒柄花苷(剂量分别为50mg/kg和200mg/kg),阳性对照组灌胃给予二甲双胍(250mg/kg),阴性对照组和模型组灌胃给予等量生理盐水,持续16周。测定大鼠血清中BUN、Scr和血糖水平,对大鼠肾脏进行病理学检查,同时采用Western Blot法检测肾组织中SOD、GSH-PX、MDA、AMPK、SIRT1、FoxO1、FN和LC3B蛋白水平。结果:模型组大鼠肾小球异常肥大,系膜细胞增殖,系膜基质增加,间质纤维化;芒柄花苷和二甲双胍治疗后,肾小球形态逐渐恢复正常,肾小球内的炎症和纤维化程度也有不同程度的改善。与阴性对照组比较,模型组、芒柄花苷低剂量组、芒柄花苷高剂量组和阳性对照组各组大鼠BUN、Scr、血糖、MDA、FoxO1和FN水平升高,SOD、GSH-PX、AMPK、SIRT1和LC3B水平降低(P<0.05)。与模型组比较,芒柄花苷低剂量组、芒柄花苷高剂量组和阳性对照组大鼠BUN、Scr、血糖、MDA、FoxO1和FN水平降低,SOD、GSH-PX、AMPK、SIRT1和LC3B水平升高(P<0.05)。与芒柄花苷低剂量组比较,芒柄花苷高剂量组和阳性对照组大鼠BUN、Scr、血糖、MDA、FoxO1和FN水平降低,SOD、GSH-PX、AMPK、SIRT1和LC3B水平升高(P<0.05)。芒柄花苷高剂量组和阳性对照组各指标差异无统计学意义(P>0.05)。结论:芒柄花苷对2型糖尿病肾病大鼠肾损伤具有明显的改善作用,其机制可能是与芒柄花苷激活AMPK/SIRT1/FoxO1通路有关。

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.

Fanlian Huazhuo Formula alleviates high-fat diet-induced nonalcoholic fatty liver disease by modulating autophagy and lipid synthesis signaling pathway

BACKGROUND Fanlian Huazhuo Formula(FLHZF)has the functions of invigorating spleen and resolving phlegm,clearing heat and purging turbidity.It has been identified to have therapeutic effects on type 2 diabetes mellitus(T2DM)in clinical application.Non-alcoholic fatty liver disease(NAFLD)is frequently diagnosed in patients with T2DM.However,the therapeutic potential of FLHZF on NAFLD and the underlying mechanisms need further investigation.AIM To elucidate the effects of FLHZF on NAFLD and explore the underlying hepatoprotective mechanisms in vivo and in vitro.METHODS HepG2 cells were treated with free fatty acid for 24 hours to induce lipid accumulation cell model.Subsequently,experiments were conducted with the different concentrations of freeze-dried powder of FLHZF for 24 hours.C57BL/6 mice were fed a high-fat diet for 8-week to establish a mouse model of NAFLD,and then treated with the different concentrations of FLHZF for 10 weeks.RESULTS FLHZF had therapeutic potential against lipid accumulation and abnormal changes in biochemical indicators in vivo and in vitro.Further experiments verified that FLHZF alleviated abnormal lipid metabolism might by reducing oxidative stress,regulating the AMPKα/SREBP-1C signaling pathway,activating autophagy,and inhibiting hepatocyte apoptosis.CONCLUSION FLHZF alleviates abnormal lipid metabolism in NAFLD models by regulating reactive oxygen species,autophagy,apoptosis,and lipid synthesis signaling pathways,indicating its potential for clinical application in NAFLD.

Forkhead box protein O1(FoxO1)regulates lipids metabolism and cell proliferation mediated by insulin and PI3K-Akt-mTOR pathway in goose primary hepatocytes

In order to explore the role of forkhead box protein O1(FoxO1)in the lipid metabolism and cell proliferation,goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVPBEZ235,and then transfected with FoxO1 interference plasmid.The related parameters of lipid metabolism and cell proliferation were measured.The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration(P<0.05);and increased the proliferative index(PI),cell DNA synthesis,protein expression of Cyclin D1 in goose primary hepatocytes(P<0.05).Secondly,the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1(P<0.05);however,there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI(P>0.05).Lastly,the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly(P<0.05).In summary,FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes.Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.

Angelica sinensis polysaccharides ameliorate 5-flourouracil-induced bone marrow stromal cell proliferation inhibition via regulating Wnt/β-catenin signaling

Chemotherapy may cause cellular oxidative stress to bone marrow.Oxidative damage of bone marrow hematopoietic microenvironment is closely related to chronic myelosuppression after chemotherapeutic treatment.Angelica sinensis polysaccharides(ASP)are major effective ingredients of traditional Chinese medicine Angelica with multi-target anti-oxidative stress features.In the current study,we investigated the protective roles and mechanisms of ASP on chemotherapy-induced bone marrow stromal cell(BMSC)damage.The human bone marrow stromal cell line HS-5 cells were divided into control group,5-FU group,5-FU+ASP group,and 5-FU+LiCl group to investigate the mechanism of ASP to alleviate 5-FU-induced BMSC proliferation inhibition.The results showed that 5-FU inhibits the growth of HS-5 cells in a time and dose-dependent manner;however,ASP partially counteracted the 5-FU-induced decrease in cell viability,whereas Wnt signaling inhibitor Dkk1 antagonized the effect of ASP on HS-5 cells.ASP reversed the decrease in total cytoplasmicβ-catenin,p-GSK-3β,and CyclinD1 following 5-FU treatment and modulated nuclear expression ofβ-catenin,Lef-1,and C-myc proteins.Furthermore,ASP also enhanced the antioxidant capacity of cells and reduced 5-FU-induced oxidative stress,attenuated FoxO1 expression,thus weakened its downstream apoptosis-related proteins and G0/G1 checkpoint-associated p27^(Kip1) expression to alleviate 5-FU-induced apoptosis and to promote cell cycle progression.All the results above suggest that the protective role of ASP in 5-FU-treated BMSCs proliferation for the chemotherapy may be related to its activating Wnt/β-catenin signaling and keeping homeostasis betweenβ-catenin and FoxO1 under oxidative stress.The study provides a potential therapeutic strategy for alleviating chemotherapeutic damage on BMSCs.

甜菜碱对糖尿病足大鼠创面愈合及AMPK/SIRT1/FoxO1通路的影响

目的探讨甜菜碱对糖尿病足大鼠创面愈合及AMPK/SIRT1/FoxO1通路的影响。方法SD大鼠随机分为对照组、模型组、二甲双胍(200 mg·kg^(-1),ig给药)组和甜菜碱低、高剂量(50、100 mg·kg^(-1),ip给药)组,每组18只。除对照组外,其余组大鼠均构建糖尿病足模型。检测大鼠空腹血糖水平和创面愈合率;ELISA法检测大鼠血清炎性因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、C反应蛋白(CRP)水平;HE染色检测大鼠创面皮肤组织病理学改变;免疫组化染色检测大鼠创面皮肤组织中CD31蛋白阳性表达;试剂盒法检测大鼠创面皮肤组织中氧化应激指标超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量;Western blotting检测大鼠创面皮肤组织中AMP依赖的蛋白激酶(AMPK)/沉默信息调节因子相关酶1(SIRT1)/叉头框蛋白1(FoxO1)通路相关蛋白表达。结果给药结束后,与对照组比较,模型组大鼠空腹血糖、血清IL-6、TNF-α、CRP水平以及创面皮肤组织中MDA含量和p-FoxO1/FoxO1值显著升高,创面愈合率和创面皮肤组织中CD31阳性表达率、SOD活性、p-AMPK/AMPK值和SIRT1蛋白表达水平显著降低(P<0.05),且创面皮肤组织病理学损伤严重;与模型组比较,甜菜碱低、高剂量组和二甲双胍组大鼠空腹血糖、血清IL-6、TNF-α、CRP水平以及创面皮肤组织中MDA含量和p-FoxO1/FoxO1值显著降低,创面愈合率和创面皮肤组织中CD31阳性表达率、SOD活性、p-AMPK/AMPK值和SIRT1蛋白表达水平显著升高(P<0.05),且创面皮肤组织病理学损伤均有不同程度改善,且甜菜碱高剂量组和二甲双胍组上述指标变化更为明显。结论甜菜碱可激活AMPK/SIRT1/FoxO1通路,减轻糖尿病足大鼠氧化应激和炎症反应,促进血管生成,加速创面愈合。

Danshensu Ameliorates Cardiac Ischaemia Reperfusion Injury through Activating Sirt1/Fox01/Rab7 Signal Pathway

Objective:To explore the specific molecular mechanisms of Danshensu(DSS)in the treatment of ischemia reperfusion injury(IRI).Methods:IRI model was established with isolated rat hearts by performing global ischaemia for 30 min,and then followed by 60 min reperfusion.Also,H9C2 cells were subjected to 4-h hypoxia followed by 3-h reoxygenation.Then 10|i mol/L DSS were added in the reperfusion/reoxygenation step to intervene IRI.Cardiac function,structural change and apoptosis were respectively tested by Langendorff System,hematoxylin and eosin(HE)and terminal-deoxynucleotidyl transferase mediated nick endabeling(TUNEL)stainings.Then lactate dehydrogenase(LDH),cardiac troponin T(cTnT),reactive oxygen species(ROS),superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX)were detected by enzyme-linked immunosorbent assay(ELISA).Sirt1/FoxO1/Rab7 Signal Pathway was monitored at both protein and mRNA levels.Results:The results showed that IRI not only greatly attenuated cardiac function(LVDP and±dp/dtmax,P<0.01,P<0.05)and increased the level of the marker enzymes(cTnT,LDH,P<0.01)from the coronary effluents,but also markedly induced changes in the structure of cardiomyocytes and contributed to apoptosis,which were mediated by boosted en doge nous ROS.However,after treatment with DSS all above indexes were improved,which was related to activating Sirt1/FoxO1/Rab7 signal pathway accompanied with the enhancement of antioxidant defense system,such as SOD and GSH-PX.Conclusion:DSS is able to protect hearts from IRI,which may be attributable to inhibiting excessive ROS through Sirt1/FoxO1/Rab7 signaling.

Ginsenoside Rb1 Protects Human Umbilical Vein Endothelial Cells against High Glucose-Induced Mitochondria-Related Apoptosis through Activating SIRT3 Signalling Pathway

Objective:To investigate whether ginsenoside Rb1(Rb1)can protect human umbilical vein endothelial cells(HUVECs)against high glucose-induced apoptosis and examine the underlying mechanism.Methods:HUVECs were divided into 5 groups:control group(5.5 mmol/L glucose),high glucose(HG,40 mmol/L)treatment group,Rb1(50μmol/L)treatment group,Rb1 plus HG treatment group,and Rb1 and 3-(1 H-1,2,3-triazol-4-yl)pyridine(3-TYP,16μmol/L)plus HG treatment group.Cell viability was evaluated by cell counting kit-8 assay.Mitochondrial and intracellular reactive oxygen species were detected by Mito Sox Red mitochondrial superoxide indicator and dichloro-dihydro-fluorescein diacetate assay,respectively.Annexin V/propidium iodide staining and fluorescent dye staining were used to measure the apoptosis and the mitochondrial membrane potential of HUVECs,respectively.The protein expressions of apoptosis-related proteins[Bcl-2,Bax,cleaved caspase-3 and cytochrome c(Cyt-c)],mitochondrial biogenesis-related proteins[proliferator-activated receptor gamma coactivator 1-alpha,nuclear respiratory factor-1 and mitochondrial transcription factor A],acetylation levels of forkhead box O3 a and SOD2,and sirtuin-3(SIRT3)signalling pathway were measured by immunoblotting and immunoprecipitation.Results:Rb1 ameliorated survival in cells in which apoptosis was induced by high glucose(P<0.05 or P<0.01).Upon the addition of Rb1,mitochondrial and intracellular reactive oxygen species generation and malondialdehyde levels were decreased(P<0.01),while the activities of antioxidant enzymes were increased(P<0.05 or P<0.01).Rb1 preserved the mitochondrial membrane potential and reduced the release of Cyt-c from the mitochondria into the cytosol(P<0.01).In addition,Rb1 upregulated mitochondrial biogenesis-associated proteins(P<0.01).Notably,the cytoprotective effects of Rb1 were correlated with SIRT3 signalling pathway activation(P<0.01).The effect of Rb1 against high glucose-induced mitochondria-related apoptosis was restrained by 3-TYP(P<0.05 or P<0.01).Conclusion:Rb1 could protect HUVECs from high glucose-induced apoptosis by promoting mitochondrial function and suppressing oxidative stress through the SIRT3 signalling pathway.

c-Abl-MST1 Signaling Pathway Mediates Oxidative Stress Induced Neuronal Cell Death

Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. We have defined that the

白藜芦醇对肌纤维类型转化的影响及调控机制研究进展

白藜芦醇作为一种来自于葡萄和其他植物的天然多酚化合物,具有抗氧化、抗癌抗肿瘤、保护心血管等作用,一直是多领域的研究热点。骨骼肌主要由肌纤维构成,肌纤维类型和组成是影响肉品质的关键因素。近年来,越来越多研究开始关注白藜芦醇对于骨骼肌纤维类型的转化,同时其具体的调控机制和相关信号通路分子逐渐被确定。本文综合国内外的相关研究报道,总结白藜芦醇对骨骼肌肌纤维类型的影响,综述microRNA、AMPK/SIRT1/PGC-1α、脂联素及FoxO1信号通路在调控肌纤维类型转化方面的作用,以期为今后靶向调控肌纤维类型转化提供理论参考。

MicroRNA-23a reduces lipopolysaccharide-induced cellular apoptosis and inflammatory cytokine production through Rho-associated kinase 1/sirtuin-1/nuclear factor-kappa B crosstalk

Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases,including sepsis.In this study,we examined the role of miR-23a in septic injury.Methods Lipopolysaccharide(LPS)was used to induce sepsis in a rat model and H9C2 and HK-2 cells.miR-23a expression was evaluated in rat myocardial and kidney tissues,as well as H9C2 and HK-2 cells.A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability,apoptosis,and the secretion of inflammatory cytokines.Furthermore,the effect of Rho-associated kinase 1(ROCK1),a miR-23a target,on cell damage was evaluated,and molecules involved in the underlying mechanism were identified.Results:In the rat model,miR-23a was poorly expressed in myocardial(sham vs.sepsis 1.00±0.06 vs.0.27±0.03,P<0.01)and kidney tissues(sham vs.sepsis 0.27±0.03 vs.1.00±0.06,P<0.01).Artificial overexpression of miR-23a resulted in increased proliferative activity(DNA replication rate:Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:34.13±3.12 vs.12.94±1.21 vs.13.31±1.43 vs.22.94±2.26,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),decreased cell apoptosis(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:11.39±1.04 vs.32.57±2.29 vs.33.08±3.12 vs.21.63±2.35,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),and decreased production of inflammatory cytokines,including interleukin-6(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:59.61±5.14 vs.113.54±12.30 vs.116.51±10.69 vs.87.69±2.97 ng/mL;P<0.05,F=12.67,HK-2 cells:68.12±6.44 vs.139.65±16.62 vs.143.51±13.64 vs.100.82±9.74 ng/mL,P<0.05,F=9.83)and tumor necrosis factor-α(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:103.20±10.31 vs.169.67±18.84 vs.173.61±15.91 vs.133.36±12.32 ng/mL,P<0.05,F=12.67,HK-2 cells:132.51±13.37 vs.187.47±16.74 vs.143.51±13.64 vs.155.79±15.31 ng/mL,P<0.05,F=9.83)in cells.However,ROCK1 was identified as a miR-23a target,and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells.Moreover,ROCK1 suppressed sirtuin-1(SIRT1)expression to promote the phosphorylation of nuclear factor-kappa B(NF-κB)p65,indicating the possible involvement of this signaling pathway in miR-23a-mediated events.Conclusion:Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1,mediated through the potential participation of the SIRT1/NF-κB signaling pathway.

假体周围骨溶解中成骨细胞自噬的信号通路

背景:最近的证据表明,自噬作为一种细胞自我保护机制,在调节成骨细胞功能和维持成骨细胞稳态方面起着重要作用,其对假体周围骨溶解的治疗与预后存在重要影响。目的:通过总结既往关于成骨细胞自噬机制的研究,为假体周围骨质溶解提供新的治疗思路和潜在的治疗靶点。方法:由第一作者应用计算机检索2015-2022年出版的文献,以“磨损颗粒、假体周围骨溶解、成骨细胞、信号通路、自噬”等为中文检索词检索中国知网、万方和维普数据库;以“wear debris,wear particles,peri^(*)prosthetic osteolysis,PPOL,Aseptic loosening,osteoblast,OB,Signal path,autophagy”等为英文检索词检索PubMed和Web of Science数据库,按照入选标准最终共纳入98篇文章。结果与结论:(1)在假体周围骨溶解中,磨损颗粒诱导的成骨细胞自噬能力的改变对于疾病的发展及转归有着关键性的作用。(2)多种信号通路共同介导成骨细胞自噬的过程,其中关键通路包括AMPK/ULK1/mTOR、核转录因子κB及Pink1/Parkin等,AMPK,mTOR及ULK1三者能够相互调节,共同维持自噬水平的稳定,核转录因子κB通路与自噬之间存在复杂的串扰,PINK1及Parkin在受损线粒体膜表面聚积,诱导线粒体自噬。(3)多条信号通路之间存在串扰,相互影响下构成了复杂的自噬网络,且在不同细胞中,相同自噬通路的激活可能会带来截然不同的影响。(4)磨损颗粒诱导的适度的自噬会减少成骨细胞的凋亡,同时增强其分化及矿化能力,改善假体周围骨溶解的预后;反之,自噬激活的不足或过度都会对成骨细胞带来损害,推动骨溶解的进展;因此,通过药物或者基因靶向调控磨损颗粒诱导的成骨细胞自噬水平可能是假体周围骨溶解治疗的方向之一。

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

黄芪多糖对糖尿病肾病大鼠氧化应激和自噬的影响及机制

目的探讨黄芪多糖(APS)对糖尿病肾病(DN)大鼠氧化应激和自噬的影响及机制。方法通过高脂饮食联合链脲佐菌素诱导DN大鼠模型,将31只建模成功的DN大鼠随机分为3组:模型组(n=11)、黄芪多糖组(n=10)和抑制剂组(n=10),另有10只未建模大鼠作为对照组。造模后,对照组和模型组大鼠灌胃2 ml生理盐水,黄芪多糖组大鼠灌胃2 ml APS[200 mg/(kg·d)]溶液,抑制剂组大鼠同时灌胃1 ml APS[200 mg/(kg·d)]溶液以及1 ml的AMP激活的蛋白激酶(AMPK)通路抑制剂Dorsomorphin[Dor,1 mg/(kg·d)]。各组大鼠均治疗12周。治疗结束后,使用强生稳步血糖仪检测空腹血糖(FPG)。通过酶联免疫吸附法(ELISA)检测空腹胰岛素(FINS),比色法检测糖化血红蛋白(GHb),CBB法检测尿蛋白、二乙酰肟比色法检测血清尿素氮(BUN)、肌氨酸氧化酶法检测尿肌酐(Cr),并计算尿白蛋白肌酐比(UACR)。使用贝克曼AU680自动生化分析仪及相应试剂盒检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)。通过WST-1法检测大鼠血清超氧化物歧化酶(SOD),钼酸铵法检测过氧化氢酶(CAT),比色法检测谷胱甘肽过氧化物酶(GSH-PX),TBA法检测丙二醛(MDA)。将肾脏切片进行苏木精和伊红(HE)染色、高碘酸-希夫(PAS)染色和Masson染色。通过qRT-PCR检测肾脏组织中AMPK、沉默信息调节因子T1(SIRT1)和叉头盒蛋白O1(FOXO1)的mRNA水平。通过Western blot检测肾脏组织中p-AMPKα、AMPKα、SIRT1、FOXO1、Acetyl-FOXO1、微管相关蛋白1轻链3B(LC3B)和P62的蛋白水平。结果与对照组比较,模型组血清FPG、FINS、GHb、UACR、BUN、TC、TG、LDL-C和MDA升高(P<0.05),血清HDL-C、SOD、CAT和GSH-PX降低(P<0.05),肾脏出现明显病变,纤维化面积升高(P<0.05),肾组织中AMPK、SIRT1和FOXO1 mRNA相对表达量降低(P<0.05),肾组织中p-AMPKα/AMPKα、SIRT1、FOXO1和LC3B的蛋白相对表达量降低(P<0.05),Acetyl-FOXO1/FOXO1和P62蛋白相对表达量升高(P<0.05)。与模型组比较,黄芪多糖组血清FPG、FINS、GHb、UACR、BUN、TC、TG、LDL-C和MDA降低(P<0.05),血清HDL-C、SOD、CAT和GSH-PX升高(P<0.05),肾脏病变明显减轻,纤维化面积降低(P<0.05),肾组织中AMPK、SIRT1和FOXO1 mRNA相对表达量升高(P<0.05),肾组织中p-AMPKα/AMPKα、SIRT1、FOXO1和LC3B的蛋白相对表达量升高(P<0.05),Acetyl-FOXO1/FOXO1和P62蛋白相对表达量降低(P<0.05)。与黄芪多糖组比较,抑制剂组血清FPG、FINS、GHb、UACR、BUN、TC、TG、LDL-C和MDA升高(P<0.05),血清HDL-C、SOD、CAT和GSH-PX降低(P<0.05),肾脏病变加重,纤维化面积升高(P<0.05),肾组织中AMPK、SIRT1和FOXO1 mRNA相对表达量降低(P<0.05),肾组织中p-AMPKα/AMPKα、SIRT1、FOXO1和LC3B的蛋白相对表达量降低(P<0.05),Acetyl-FOXO1/FOXO1和P62蛋白相对表达量升高(P<0.05)。结论APS通过调节DN大鼠肾组织中AMPK/SIRT1/FOXO1信号通路激活自噬并抑制氧化应激,从而改善DN大鼠糖脂代谢并减轻肾损伤。

A soy glycinin derived octapeptide protects against MCD diet induced non-alcoholic fatty liver disease in mice

Soy glycinin derived octapeptide(SGP8)is a peptide obtained from degradation of the soy glycinin,whose amino acid sequence is IAVPGEVA.To determine the effect of SGP8 on non-alcoholic fatty liver disease(NAFLD),steatosis Hep G2 cells were induced by 1 mmol/L free fatty acid(FFA)and C57 BL/6 J mice were fed with methionine-choline defi cient(MCD)diet for 3 weeks to establish NAFLD model.The results of oil red O staining and total cholesterol(TC)/triglyceride(TG)contents showed that SGP8 could signifi cantly reduce the lipid content of steatosis Hep G2 cells.In vivo,SGP8 lowered plasma alanine aminotransferase(ALT)and low density lipoprotein(LDL)content,normalized hepatic superoxide dismutase(SOD)and malondialdehyde(MDA)production,and reduced the severity of liver infl ammation.The results of Western blotting showed that SGP8 increased expression of Sirtuin-1(SIRT1)and phosphorylation level of AMP activated protein kinase(AMPK)in hepatocytes.Through activation of SIRT1/AMPK pathway,SGP8 downregulated the expression of sterol regulatory element binding protein 1 c(SREBP-1 c)and its target genes ACC and FAS expression levels,and increased the phosphorylation level of acetyl Co A carboxylase(ACC).Furthermore,SGP8 also upregulated the expression of transcription factor peroxisome proliferator activated receptorα(PPARα),which was regulated by SIRT1/AMPK pathway,and its target gene CPT1 level.In conclusion,SGP8 might improve NAFLD by activating the SIRT1/AMPK pathway.Our data suggest that SGP8 may act as a novel and potent therapeutic agent against NAFLD.