| Hyperlipeidmia characterized by elevated levels of total cholesterol and triglycerides in the blood is one of the important risk factors for cardiovascular and cerebrovascular diseases.But the clinical symptoms of hyperlipeidmia are not obvious,it will lead to various metabolic disorders if not treated in time.Hyperlipeidmia is closely associated with genetic or dietary factors.The long-term excessive intake of high-cholesterol and high-fat foods will significantly increase the risk of hyperlipeidmia.Currently available drugs for treating hyperlipeidmia have significant side effects and are expensive,making it urgent to develop new treatment strategies.Lysimachia christinae is a traditional Chinese herb,and its extract has various pharmacological effects such as antioxidant,anti-inflammatory,hepatic protection,and lipid-lowering.Polysaccharides are one of the main active components of natural plants,with complex structures and various biological activities.In addition,natural plant polysaccharides have also been widely studied as regulators of gut microbiota.Using natural plant polysaccharides as gut microbiota regulator is a new direction to develop drugs for hyperlipeidmia.In this study,Lysimachia christinae polysaccharide was extracted,purified and characterized by gel permeation chromatography,high performance liquid chromatography,scanning electron microscope and fourier transform infrared spectroscopy.The biological activity of polysaccharide in vitro was explored by lithocholic acid-induced cell injury model,lipopolysaccharide-induced inflammation model,and mouse spleen lymphocytes.Then hyperlipeidmia mouse model that induced by high-fat diet was used,the lipid-lowering effect of polysaccharide was studied by means of Western blot and q PCR.Later,gut microbiota sequencing,metabonomics,and fecal microbiota transplantation were used to further explore the mechanism of polysaccharide in the treatment of hyperlipeidmia.Finally,several rounds of animal experiments were carried out by using genetic engineering bacteria and adeno-associated virus-mediated gene editing techniques to further verify the mechanism of polysaccharide in the treatment of hyperlipeidmia.The main results are as follows:1.Purification,characterization and bioactive activity of polysaccharide from L.christinae in vitro.Natural polysaccharide(NP)was isolated and purified from L.christinae,then its structure and biological activities were identified.The average molecular weight of NP was 38.7 k Da.NP was mainly composed of glucose,mannose,arabinose,and glucuronic acid,with glucose accounting for more than 50%.NP exhibited protective effects against lithocholic acid-induced damage in L02,Hep A,and Caco-2 cells,indicating it possesses powerful antioxidant and anti-apoptotic abilities.In a lipopolysaccharide-induced macrophage inflammation model,NP inhibited the production of inflammatory mediators,including NO,TNF-α,IL-6,and IL-1β.Meanwhile,NP stimulated the proliferation of mouse lymphocytes,especially CD3~+CD4~+T cells,and significantly promoted the production of anti-inflammatory cytokine IL-10.2.Study on the therapeutic effect of L.christinae polysaccharides on hyperlipeidmia mice.A high-fat diet,also known as lithogenous diet(LD),was uesd to establish a hyperlipeidmia mouse model.In the model group,the contents of total cholesterol,triglyceride,inflammatory factors IL-1βand IFN-γwere significantly increased,while the activity of SOD and CAT,and the content of GSH and IL-10 in the serum were decreased,and the proportion of CD3~+CD4~+T cells in the spleen was decreased.Oral administration of NP significantly reduced blood lipid levels,inhibited oxidative stress,alleviated organ damage,and reduced inflammation in the mice,demonstrating the protective effects of NP in hyperlipidemic mice.Futhermore,NP promoted CYP7A1 protein expression in the liver and inhibited the intestinal FXR-FGF15 signaling pathway.However,cell experiments showed that NP could not directly affect the expression of related proteins,suggesting that NP may regulate this signal pathway through other ways.3.Effect of L.christinae polysaccharides on gut microbiota in hyperlipidemic mice.NP has been shown to have various benefits for cholesterol metabolism,but its molecular mechanism remains unclear.To investigate whether NP could regulate FXR-FGF15 pathway by regulating the gut microbiota,mice were fed LD and intervened with NP.Next,we analysed the changes in gut microbiota composition through high-throughput sequencing and measured the changes of bile acid metabolism in the ileum using liquid chromatography-mass spectrometry.The results showed that NP altered the changes in gut microbiota and bile acid metabolism induced by LD,significantly reducing the levels of bacteria such as Lactobacillus murinus and Escherichia coli in LD mice.In addition,NP reduced the levels of unconjugated bile acids and increased the levels of conjugated bile acids in the ileum,consistent with changes of ileal bile salt hydrolase(BSH)activity.Further,fecal microbiota transplantation experiments confirmed that NP could improve hyperlipeidmia by regulating the intestinal flora and could also affect the FXR-FGF15 signaling pathway.4.Effects of BSH from Lactobacillus on hyperlipeidmia mice.To verify the role of L.murinus and its BSH,we isolated L.murinus from the ileal contents in mice and transferred its BSH genes to Brevibacillus choshinensis through genetic engineering.The engineered bacteria were then orally administered to mice fed a high-fat diet.The results showed that intervention with L.murinus and its BSH significantly increased the blood lipid levels in mice,indicating that L.murinus and its BSH are key factors in the elevated blood lipids in mice.The changes of bile acid metabolism caused by BSH in model mice activated the FXR-FGF15 signaling pathway in the ileum,thereby inhibiting the CYP7A1 protein expression in the liver.5.Effects of FGF15 gene overexpressing or silencing on cholesterol metabolism in mice.To verify the role of the FXR-FGF15 signaling pathway in hyperlipeidmia,we established mouse models that overexpressing or inhibiting of FGF15 mediated by adeno-associated virus(AAV).The results showed that overexpression of FGF15increased the blood lipid levels in mice fed a normal diet,and silencing of FGF15significantly decreased the blood lipid levels in mice fed a high-fat diet.In addition,changes in the expression of FGF15 further affected the expression of CYP7A1 in the liver,indicating that FGF15 can regulate the expression of CYP7A1 and is an important target for the treatment of hyperlipeidmia.To sum up,in this paper,the polysaccharide NP was extracted and purified,and proved that NP has strong antioxidant,anti-inflammatory,and immunomodulatory ability in vitro and in vivo.This study confirmed that NP can improve hyperlipeidmia induced by high-fat diet in mice.Based on the multi-omics analysis of metabonomics and microbiome,the regulatory effect of NP on gut microbes and bile acid metabolism was clarified.In addition,this study also found a signature differential strain L.murinus in hyperlipemic mice,and clarified that BSH produced by L.murinus is one of the factors leading to hyperlipeidmia,mainly by increasing the content of unconjugated bile acids in the ileum to active the FXR-FGF15 pathway,thus inhibiting the expression of CYP7A1 in the liver,and finally promoting the occurrence of hyperlipeidmia by inhibiting the transformation of cholesterol to bile acids in the liver.This study provides an important direction and lays a solid foundation for the development of therapeutic drugs for hyperlipeidmia based on natural plant polysaccharides. |
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