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.

Mechanism of Profilin-1 in regulating eNOS/NO signaling pathway and its role in hypertensive myocardial hypertension

Objective:To explore the mechanism of Profilin-1 in regulating eNOS/NO pathway and its role in the development of myocardial hypertrophy.Methods:Spontaneously hypertensive rats(SHR) aged 5 weeks were injected with different adenovirus vectors to induce Profilin-1expression knockdown(SHR-I) or over express(SHR-H) or to use as control(SHR-C).All these treatment were compared with Wistar-Kyoto rats(SKY) treated with control adenovirus vectors(WKY-C).The same injection was executed at the sixth week during the experiment of 12 weeks.After experiment,the left ventricular weight-to-heart weight ratio(LVW/HW)and left ventricular long axis(LVLA) were measured.Meanwhile.NO contents in blood and myocardium,Profilin-1,eNOS and Caveolin-3 niRNA and protein levels and phosphorylated eNOS(P-eNOS) protein level in myocardium were determined.Results:Compared with WKY-C group,the SHR-C group was statistically higher in LVW/HW(0.79±0.03).LVLA(11.82±0.58 mm) and Profilin-1 niRNA and prolein level(P<0.05).but lower in NO content[(18.63±6.23) μmol/L| in blood and[(2.71±0.17) μmol/L]in myocardium).eNOS activity and Caveolin-3 expression(P<0.05).The over expressing Profilin-1 led SHR-H group to a higher value of LVW/HW[(0.93±0.03) mm and LVLA(14.17±0.69) mm]in comparison with SHR-C group(P<0.05).and to a lower value of NO content(in myocardium).eNOS activity and Caveolin-3 expression(P<0.05):however,this phenomenon was reversed by the knockdown Profilin-1 expression(SHR-I group).Conclusions:Profilin-1 expression,being negative in regulating Caveolin-3 expression and eNOS/NO pathway activity,promotes the development of myocardial hypertrophy which can be reversed by Profilin-1 silencing.

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.

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.

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.

Protective Effect of SGLT2 Inhibitor on D-Galactose-Induced Senescence in Mice and Its Mechanism

Objective: To observe the cerebral protective effect of dagliflozin, a sodium-glucose co-transport protein 2 (SGLT2) inhibitor, in aging mice and to explore its molecular mechanism. Methods: 1. 66 male C57BL/6 mice were divided into control group (13) and model group (53), and the model group was moulded by subcutaneous injection of D-galactose into the back of the neck, while the control group was treated with equal amount of saline for 8 weeks. The weight of each group of mice was observed and recorded every 7 days, and two groups of mice were randomly selected for frozen sections of brain tissue at the end of the modelling period to verify the aging model. 2. After the aging model was successfully established, the aging groups were divided into 5 groups: model group, dagliflozin-treated group (high and low dose), and dagliflozin + ex527-inhibited group (high and low dose). Fasting blood glucose was measured in each group every 2 weeks for 8 weeks. At the end of treatment, Morris water maze was performed at the end of the treatment. After execution of the mice, the organ indices of heart, brain, liver, kidney and spleen were measured;the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were determined. Results: After the successful establishment of the aging model, it was found that during the treatment phase of dagliflozin. 1) The organ indices of mice in the aging group were significantly lower than those of other groups, and no significant hypoglycemia was observed throughout the treatment process. 2) In the water maze test, mice in the aging group had a significantly longer latency in the plateau phase compared to the control and treatment groups, while the number of times the mice crossed the original plateau and the percentage of time spent exploring the original plateau quadrant were reduced after the plateau was removed. 3) The nerve cells in the aging mice were disorganized and the nuclei of the mice were deeply stained;the dagliflozin group improved the morphological changes in the brain of aging mice. 4) In addition, compared with the control mice, the serum MDA level was significantly increased and the antioxidant enzyme SOD activity was significantly decreased in the aging group, while compared with the aging group, dagliflozin significantly decreased the MDA level and increased the SOD activity. 5) The expression of SIRT1 and PGC-1α was significantly upregulated in the low and high doses of dagliflozin compared to the aging group. Conclusion: The present study suggests that dagliflozin can delay organ aging, improve the learning and memory ability of aging mice, and exert antioxidant effects, probably through upregulating the SIRT1/PGC-1α signaling pathway.

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.

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.