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.

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.

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.

Solanine Interferes with AKT/p-AKT and PI3K/p-PI3K Pathway to Inhibit HIF and Destroy Cell Energy Metabolism

The purpose of this study was to explore the mechanism of Solanine disrupting energy metabolism in human renal cancer ACHN cells and to clarify its target. The specific method was to culture human renal cancer ACHN cell lines, and to intervene with Solanine of high, medium and low concentrations. The content of ATP in cells was measured by ELISA method. The expression of HIF-1α protein and the expression of PI3K, AKT, p-PI3K, p-AKT in PI3K/AKT pathway were detected by Western blotting. The results showed that compared with the control group, the relative expression of p-PI3K and p-AKT showed a downward trend with the increase of Solanine concentration (P < 0.05), while the relative expression of PI3K and AKT showed no significant change (P > 0.05). In addition, the relative expression of HIF-1α also showed a downward trend (P < 0.05). According to the above results, it is suggested that Solanine can significantly inhibit the energy metabolism of renal cancer cells, the main mechanism of which is the down-regulation of HI-1αf downstream of the PI3K/Akt pathway by inhibiting the phosphorylation process of PI3K/p-PI3K and Akt/p-Akt.

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.

Promotion and Mechanism of Acupotomy on Chondrocyte Autophagy in Knee Osteoarthritis Rabbits

Objective:To explore the effect of acupotomy intervention on autophagy of chondrocytes in rabbits with knee osteoarthritis(KOA),and to determine the possible mechanisms of acupotomy to alleviate cartilage degeneration.Methods:The modified Videman method was used to construct a KOA rabbit model.After modeling,40 rabbits were randomly divided into 4 groups by a random number table:control;KOA(model);KOA+acupotomy(acupotomy),and KOA+sham acupotomy(sham),10 in each group.After a 3-week treatment course,the knee joint activity was determined by the modified Lequesne MG index.Hematoxylin-eosin staining staining was used to examine the morphological changes of chondrocytes.Autophagy of chondrocytes was observed by transmission electron microscopy.The surface morphology of cartilage tissue was observed by scanning electron microscope.The mRNA and protein levels of AMP kinase/mammalian target of rapamycin/Unc-51(AMPK/mTOR/ULK1)signal pathway key proteins,autophagy-related factor Beclin-1 and microtubule-associated protein 1A/1B light chain 3(LC3)in rabbit knee cartilage were assessed by real-time fluorescence quantitative polymerase chain reaction and Western blot,respectively.Results:The modified Lequesne MG score of acupotomy group was significantly lower than that of model group(P<0.05).Pathological results showed that chondrocyte autophagy decreased and cartilage surface was rough in the model group,which recovered after acupotomy treatment.The mRNA expressions of AMPK,ULK1,Beclin-1 and the protein levels of p-AMPK,p-ULK1,Beclin-1,and LC3Ⅱ/LC3Ⅰwere decreased in the model group,while the mRNA and protein expressions of mTOR were increased(P<0.01).However,acupotomy treatment reversed these abnormal changes(P<0.05).Conclusions:Acupotomy could effectively up-regulate the expressions of AMPK,ULK1 and Beclin1,reduce the expression of mTOR,promote autophagy,and alleviate joint degeneration.Acupotomy is a promising complementary and alternative therapy for KOA.

Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-inducedferroptosis

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide.Influenza A virus(IAV)has been found to activate multiple programmed cell death pathways,including ferroptosis.Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation,leading to cell death.However,little is known about how influenza viruses induce ferroptosis in the host cells.In this study,based on network pharmacology,we predicted the mechanism of action of Maxing Shigan decoction(MXSGD)in IAV-induced ferroptosis,and found that this process was related to biological processes,cellular components,molecular function and multiple signaling pathways,where the hypoxia inducible factor-1(HIF-1)signaling pathway plays a significant role.Subsequently,we constructed the mouse lung epithelial(MLE-12)cell model by IAV-infected in vitro cell experiments,and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage,increased reactive oxygen species(ROS)release,increased total iron and iron ion contents,decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4(GPX4),increased expression of acyl-CoA synthetase long chain family member 4(ACSL4),and enhanced activation of hypoxia inducible factor-1α(HIF-1α),induced nitric oxide synthase(iNOS)and vascular endothelial growth factor(VEGF)in the HIF-1 signaling pathway.Treatment with MXSGD effectively reduced intracellular viral load,while reducing ROS,total iron and ferrous ion contents,repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway.Finally,based on animal experiments,it was found that MXSGD effectively alleviated pulmonary congestion,edema and inflammation in IAV-infected mice,and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.

Extracts of Celastrus Orbiculatus Inhibit Cancer Metastasis by Down-regulating Epithelial-Mesenchymal Transition in Hypoxia-Induced Human Hepatocellular Carcinoma Cells

Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide(MTT), scratch-wound and transwell assays, respectively. Co Cl2 was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immuno?uorescence analysis,respectively. Results: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner(P<0.01). Furthermore, the expression of epithelial-mesenchymal transition(EMT) related markers were also remarkably suppressed in a dose-dependent manner(P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole(YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced Hep G2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect(P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.