Intervention Effect Of Exercise With Lycium Ruthenicum Murr Active Constituent Compliment On NAFLD Mice

Research background and purposeIn clinical practice,an initial diagnosis of NAFLD is usually established with radiological imaging techniques,by the presence of?5%hepatic fat accumulation in the absence of other recognized causes of fatty liver,e.g.,alcohol,virus,drugs,autoimmunity.In simple steatosis,only hepatic steatosis is observed and this phase is reversible.The main influencing factors of lipid accumulation in liver include lipid synthesis,oxidation of fatty acids and oxidative stress during the oxidation of fatty acids.The incidence of NAFLD/NASH has been rapidly increasing globally in line with the increased prevalence of obesity and reached 17%-33%,and is currently the most common chronic liver disease.Recently,the incidence of NAFLD and NASH was reported to be 46%and 12%,respectively,in a largely middle-aged population.However,the pathogenesis of NAFLD/NASH has not been completely elucidated,and there is a lack of consensus regarding the most useful and appropriate pharmacological therapy.Currently,the principal treatment for NAFLD/NASH is lifestyle modification by diet and exercise.Exercise can reduce lipid content and markers of hepatocyte injury in NAFLD patients.This protective mechanism includes powerful changes in blood lipid and lipoprotein content,insulin metabolism,liver fibrosis and inflammation,etc.However,there are few cellular and molecular mechanisms that aerobic exercise promote the benign outcome of NAFLD.The motherland medicine believed that Lycium Ruthenicum Murr even,taste sweet,and rich in protein,lyceum barbarum polysaccharide,amino acids,vitamins,alkaloids,minerals,trace elements and other nutrients,other more,it has abundant Pigment of Lycium Ruthenicum Murr-natural proanthocyanidins,which has the function of scavenging free radicals and anti-oxidation.Modern medical research has confirmed that the active constituent of Lycium Ruthenicum Murr can improve the anti-fatigue ability of mice,reduce blood glucose,promote the ability of radical scavenging,reduce hyperlipidemia and lipid per-oxidation.And the effectiveness and mechanisms of aerobic exercise combined with the active constituent of Lycium Ruthenicum Murr need more systematically studies.The active ingredients of black fruit lycium barbarum mainly include polysaccharides,pigments and flavonoids.We explore the intervention effectiveness,linkage effectiveness and the possible mechanism of NAFLD which intervene by aerobics combine with the active constituents of Lycium Ruthenicum Murr complement through detection the relative gene expression of lipid Deno synthesis and fatty acid oxidation involved pathways and the oxidative stress in NAFLD mice,who intervene by aerobic exercise,polysaccharides,pigments,flavonoids and aerobic exercise combine with polysaccharides,aerobic exercise combine with pigments,aerobic exercise combine with flavonoids.H₂S,as a novel endogenous gaseous signal transducer?gasotransmittor?,is synthesized endogenously during cysteine metabolism via three enzymes including3-MST,CBS and CSE,and the CSE accounts for most of H₂S formation in liver.The emerging role of H2S in multiple facets of inter-and intra-cellular signaling as a metabolic,anti-hypertensive,anti-inflammatory and anti-oxidant molecule,inflammatory,neuro and vascular modulator has been increasingly realized.The studies about the effect of exercise to H₂S signaling molecules,a few articles mainly focus on myocardial endoplasmic reticulum stress,pulmonary fibrosis pathways,and chronic aerobic exercise training restored endogenous H2S formation and improved autophagy influx in livers.Recent studies have found that H2S regulating the SREBP-1c signaling pathway to affect liver synthesis and which is consistent with the possible pathway of aerobic exercise to intervene NAFLD.So,we speculated that aerobic exercise may play an important regulatory role in the synthesis of endogenous H₂S in the liver of NAFLD mice,and the regulation of lipid synthesis by influencing the content of H₂S is one of main mechanisms for the intervention of aerobic exercise in NALFD.In order to verify the guess,we explore the mechanism of aerobic exercise promote postoperatively of NAFLD by detection the content of H2S,the expression of regulate genes of H2S and the relative expression of the SREBP-1c pathway in liver of NAFLD which intervention alone with aerobic exercise,liver generate H₂S key genes CSE inhibitors PAG,exogenous H₂S supplements NAHS and intervention by aerobic exercise combined with PAG,aerobic exercise combined with NAHS intervention NAFLD.MethodsPART 1:Established the NAFLD models induced by high sugar and high fat dietNutrition-induced animal model?food feeding?is the most commonly used method for establish NAFLD models,in this study,we establish NAFLD models by perfusion of high sugar and high fat diet emulsion,and observe the pathological damages in liver and the change of relative indexes about the function of liver.PART 2:The intervention effects of 7 weeks aerobic exercise combined with Lycium Ruthenicum Murr.active constituent on NAFLD MiceNAFLD mice intervene to aerobic exercise,polysaccharides,pigments,flavonoids alone and aerobic exercise combine with polysaccharides,aerobic exercise combine with pigments,aerobic exercise combine with flavonoids.Observe the pathological damages in liver,the change of relative indexes about the function of liver and oxidation stress and the Lipid deposition in the liver.PART 3 The study of key signal transduction pathway related lipid Deno synthesis of NAFLD which intervention to 7 weeks aerobic exercise combined with Lycium Ruthenicum Murr.active constituentNAFLD mice intervene to 7 weeks of aerobic exercise,polysaccharides,pigments and flavonoids,aerobic exercise combine with polysaccharides,aerobic exercise combine with pigments,aerobic exercise combine with flavonoids,Observe the relative gene and protein expression of SREBP-1c pathway.PART 4 The study of key signal transduction pathway related fatty oxidation of NAFLD which intervention to 7 weeks aerobic exercise combined with Lycium Ruthenicum Murr.active constituent.NAFLD mice intervene to 7 weeks of aerobic exercise,polysaccharides,pigments and flavonoids,aerobic exercise combine with polysaccharides,aerobic exercise combine with pigments,aerobic exercise combine with flavonoids,Observe the relative gene and protein expression of PPAR?signal pathway and the rate of?-oxidation of mitochondria.PART 5 The influence of aerobic to the content of H2S and the mechanism of aerobic intervene lipid Deno synthesis in NAFLD.NAFLD mice intervene to 7 weeks of aerobic exercise,liver generate H₂S key genes CSE inhibitors PAG,exogenous H₂S supplements NAHS alone and intervention to aerobic exercise combined with PAG,aerobic exercise combined with NAHS intervention NAFLD.Observe the pathological damages in liver,the Lipid deposition in the liver,the content of H₂S in liver and peripheral blood,the expression of CSE and CBS,the relative gene and protein expression of SREBP-1c signal pathway.ResultsPART 1:Established the NAFLD models induced by high sugar and high fat diet1.General conditions:NAFLD model mouse have higher body weight,liver weight and liver index compare to normal KM mouse.2.Pathological examination:The microscopic structure showed that in normal group,hepatica lobule structure is organized,the size of liver cells is even,with abundance of lipid droplet vacuoles,The microscopic structure showed that in MX group,hepatic lobule structure is disorganized,with nucleus abnormal and surrounding cells,and there were a large number of lipid droplet vacuoles in hepatocytes,in intervene groups,hepatic lobular had clear structure,the number of intracellular lipid droplets significantly decreased.3.The index of liver damage:compare to KB,the activity of ALT?AST,the content of TG?TC?LDL-c is significantly increased in the serum of MX,while the content of HDL-c is significantly decreased.PART 2:The intervention effects of 7 weeks aerobic exercise combined with Lycium Ruthenicum Murr.active constituent on NAFLD Mice1.General conditions:Compare with MX group,the body weight have no significantly change in the group of P?C?F,while the liver weight significantly decreased;the body weight,liver weight and liver index are significantly decreased in E?EF?EC?EP?P<0.05 or P<0.01??2.Pathological examination:Compare with MX group,the pathological damage is significantly decreased in every intervene group.3.Relative indicators of lipid deposition:Compare with MX group,the content of TG?TC is significantly decreased in the liver of intervene group,there are synergistic effect of the combine intervention of aerobic exercise and the active constituents of LRM on the content of TG and TC.4.Relative indicators of liver damage:Compare with MX group,the activity of AST?ALT,the content of LDL-c are significant decreased,while the content of HDL-c is significant increased in all intervene group?P<0.01?.To the activity of AST,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides.To the content of HDL-c,there are synergistic effect with combine intervene in aerobic exercise and polysaccharides,pigments,flavonoids.To the content of LDL-c,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides,pigments.5.Relative indicators of oxidative stress:Compare with MX group,the content of TBARS?GSH and the activity of SOD are significantly increased in all intervene group.To the activity of SOD and the content of GSH,there is a synergistic effect with combine intervene in aerobic exercise and pigments,flavonoids.To the content of TBARS,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides,pigments.PART 3 The study of key signal transduction pathway related lipid Deno synthesis of NAFLD which intervention to 7 weeks aerobic exercise combined with Lycium Ruthenicum Murr.active constituentRelative expression of SREBP-1c signal pathway:Compare with MX group,the gene and protein expression of LXR-alpha?SREBP-1c and TNF-alpha are significantly decreased in intervene group,while the gene expression of target genes of ACC?FAS?SCD1 are significantly decreased.There have no change of Insig-1 in C and EC group,the gene and protein expression of Insig-1 are significantly decreased in P and EP,the gene expression of Insig1 have no change in F,while the protein expression is decreased.To the gene expression of LXR-alpha,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides,flavonoids.To the gene expression of ACC,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides,flavonoids,pigments.To the gene expression of SCD1,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides.To the ptotein expression of TNF-alpha,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides,flavonoids,pigments.PART 4 The study of key signal transduction pathway related fatty oxidation of NAFLD which intervention to 7 weeks aerobic exercise combined with Lycium Ruthenicum Murr.active constituent.1.?-oxidation in Mitochondria:mitochondria are main place to fatty acid oxidation and regulate by PPAR alpha signal pathway,CPT1 and LCAD are downstream target genes which influence the condition of?-oxidation in mitochondria.Compare with MX group,the gene and protein expression of PPAR alpha and the gene expression of CPT1 and LCAD have no change in P group,the gene and protein expression of PPAR alpha and the gene expression of CPT1 and LCAD are significantly increased in E?EP?EC?EF?C?F group.To the gene and protein expression of PPAR alpha and the gene expression of LCAD,there is a synergistic effect with combine intervene in aerobic exercise and polysaccharides.2.?-oxidation in peroxisome and?-oxidation in microsome are secondary metabolic places,CYP2E1?CYP4A10 and CYP4A12 are key enzymes to?-oxidation in microsome and AOX is rate limiting enzyme to?-oxidation in peroxisome,the genes which encoding above enzymes are regulate by PPAR-alpha.Compare with MX group,the gene and protein expression of CYP2E1 and the gene expression of AOX?CYP4A10?CYP4A12 have no change in P group and significantly increased in other intervene groups.To the gene expression of CYP4A10,there is a synergistic effect with combine intervene in aerobic exercise and pigments.3.The rate of.?-oxidation in mitochondria:Compare with MX group,the rate of.?-oxidation in mitochondria significantly increased in all intervene groups,and there is synergistic effect with combine intervene in aerobic exercise and polysaccharides,pigments.PART 5 The influence of aerobic to the content of H2S and the mechanism of aerobic intervene lipid Deno synthesis in NAFLD.1.The influence of PAG in the generate of H₂S and the expression of SREBP-1c signal pathway:The gene expression of CSE and the content of H₂S in liver and serum are significantly decreased and the gene expression of CBS and SREBP-1c signal pathway are significantly increased in liver with HFD diet.The overexpression rate of CSE gene is about-73.43%in mouse liver by CSE inhibitor.And PAG have no change in body weight and the gene expression of CBS,but significantly increased liver wet weight,liver index,TG content in liver.HE staining and Ultra-micro electron microscopy showed further liver injury.Compare with Pa group,EPa group have better performance in the gene expression of CBS,the liver index,the content of H₂S in liver and significantly decrease the gene expression of SREBP-1c signal pathway but still show poor performance compare with E.To the liver index,the content of H₂S in liver and the gene expression of FAS,there is antagonistic effect to aerobic exercise and PAG,to the body weight,there is synergistic effect to aerobic exercise and PAG.2.The influence of exogenous H₂S supplements NAHS in the generate of H₂S and expression of SREBP-1c signal pathway:The gene expression of CSE and CBS have no change for the NAHS intervene NAFLD mouse,while the body weight,liver index,the content of TG,the damage of liver and the gene expression of SREBP-1c are significantly decreased,and the content of H₂S in liver and serum is increased.Compare with E and Na group,ENa have lower weight,liver index,the content of TG,the expression of SREBP-1c and higher content of H₂S.To the content of H₂S in liver,the gene expression of SREBP-1c and FAS,there is synergistic effect to aerobic exercise and NAHS.ConclusionThis study explores the effects and possible regulatory pathways of aerobic exercise combined with Lycium Ruthenicum Murr.active constituent,other more,we trying to find the molecular mechanism of aerobic exercise intervention NAFLD.1.7 weeks aerobic exercise and Lycium Ruthenicum Murr.active constituent interfered the lipid synthesis of NAFLD by regulation the SREBP-1c signal pathway.Effects of aerobic exercise combined the active components of fructus melanoglycium barbarum is better than that of single intervention.2.The effect of polysaccharides interfere fatty acid oxidation is limited than that of aerobic exercise?pigments and flavonoids.Effects of aerobic exercise combined the active components of fructus melanoglycium barbarum is better than that of single intervention.3.The lipid deposition injury in liver which induced by high sugar and high fat diet is related to H S signal transduction,aerobic exercise and NAHS can repair the injury,while PAG have opposite effect.The mechanism of aerobic exercise intervene lipid synthesis is related to SREBP-1c signal pathway.