| Eupolyphaga steleophaga(Corydi-idae)is the dry body of female insect of Eupolyphaga sinensis Walker or Steleophaga plancyi Bolen.It has functions of activating meridians,breaking blood and swelling.Because of the complex chemical composition,its pharmacological action is extensive.With the development of science and technology,the fields of application of Eupolyphaga steleophaga are gradually expanded,and the researchs on pharmacological effects are gradually thorough.A large number of studies and clinical results have shown that Eupolyphaga steleophaga can cure cancer,regulate lipid metabolism and eliminate free radicals,and can treat and prevent a variety of diseases.The further study will contribute to reveal the drug action pathway and related mechanism,and provide theoretical basis and research direction for full use of the curative effect.Hypercholesterolemia is a kind of dyslipidemia disease,which characteristic is a higher serum total cholesterol(TC)than the normal.The disease can lead to peripheral vascular diseases,induced myocardial infarction,stroke,and stroke.Because of serious damage to human health,the disease prevention and treatment should be highly valued.Rabbits are one of the ideal models for the study of hypercholesterolemia.Because rabbits are sensitive to cholesterol,high cholesterol feeds can induce hypercholesterolaemia in these model animals in the short term.Rabbits not only contribute to the pharmacological and pathological studies of hypercholesterolemia in humans,but also facilitate the transformation of research results.At present,the drugs for the prevention and treatment of hypercholesterolemia are mainly western drugs,mostly single-target,with strong side effects.The anti-cholesterol effect of Eupolyphaga steleophaga has the advantages of multiple targets and small adverse reactions.Sixtiy male Harbin White rabbits(Leporidae)were fed a normal feed(n=15)or a high cholesterol feed(n=45)for 8 weeks to induce hypercholesterolaemia.After the model establishment,hypercholesterolemia rabbits were randomly divided into 3 groups,including 11 rabbits of control group and 12 rabbits of high-dose and low-dose treatment groups,respectively.The high-dose and low-dose treatment groups were fed standard diets containing 0.4g·kg-1 and 0.2g·kg-1,respectively.The blank group and the control group were fed standard diets for 6 weeks.The total cholesterol(TC)levels of rabbits in different groups were detected to establish the model.GeNorm,BestKeeper,NormFinder,?Ct and RefFinder algorithms were used for evaluation the expression stability of 9 commonly used reference genes in adrenal gland,liver,spleen and kidney.Fluorescence quantitative PCR and western-blot were used to analyse the expression changes of seven genes(LDLR,LXRa,ABCA1,SREBP-2,HMGCR,FXR? and CYP7A1)in the liver of each group,respectively,for exploreing the anti-hypercholesterolemia mechanism of Eupolyphaga steleophaga.Serum AST,ALT and AST/ALT levels were detected to evaluate the liver damage caused by Eupolyphaga steleophaga at low and high doses.The main research results are as follows:1)At the end of the modeling rabbits,the TC level of the hypercholesterolemia rabbit group was significantly higher than that of the blank group(P<0.01),indicating that Harbin white rabbits could be the model animal of hypercholesterolemia.At the end of the treatment,control group TC were significantly higher than that of blank group and high and low dosage of medicine group(P<0.01).There were no significant differences between the high and low dosage groups(P>0.05),showed that the two doses had the same cholesterol-regulating effect.2)In the anti-hypercholesterolemia model of Eupolyphaga steleophaga,the stability of nine reference genes evaluated by GeNorm,BestKeeper,NormFinder.A Ct and RefFinder in four different tissues are as follows:in the adrenal glands,four relatively stable reference genes:Hprt1,B2m,Actb and Eeflal,two most unstable reference genes:Gapdh and Sdha,and the optimal reference genes:Hprtl(GM=1.968)and B2m(GM=2.968);in the liver,four relatively stable reference genes:Gapdh,B2m,Ywhaz and Eef1al,three most unstable reference genes:Hprtl,Actb and Rpl5,and the optimal reference genes:Gapdh(GM=2.236)and B2m(GM=2.449);in the spleen,four relatively stable reference genes:Gapdh,Ywhaz,Rpl5 and Sdha,two most unstable reference genes:Eeflal and Actb,and the optimal reference genes:Gapdh(GM=2.060)and Ywhaz(GM=2.340);in the kidney,four relatively stable reference genes:Ppia,B2m,Gapdh and Actb,two most unstable reference genes:Rpl5 and Eef1al,and the optimal reference genes:Ppia(GM-1.968),B2m(GM=2.060)and Gapdh(GM=2.449).3)The expression of Pparyl were normalized by Gapdh?B2m and Actb,respectively.The trends of relative expression were consistent among the four different groups by Gapdh.And the the trends of relative expression of B2m were slightly different among the groups.But the the trends of relative expression of Actb were varied greatly among groups.So Gapdh and B2m were more suitable for model than Actb.4)At the end of treatment,the results showed that LDLR gene expression was significantly up-regulated at the level of transcription and translation in the high-dose and low-dose groups compared with the control group(P<0.05),indicating that Eupolyphaga steleophaga could enhance LDLR gene expression and promote LDL absorption.Compared with control group,high and low doses group LXRa expressed in transcription and translation level was significantly up-regulated?P<0.05),the ABCA1 expression of high does was significantly up-regulated at transcription and translation levels(P<0.05),the ABCA1 expression of low does group was significantly up-regulated at transcription level(P<0.05)and had no significant difference at translation level(P>0.05),and these results indicated that Eupolyphaga steleophaga couled promote liver cell cholesterol efflux and reduce cholesterol level in the liver cells,and high dose was more effective.Compared with control group,the SREBP-2 expression of high doses group was significantly lowered at transcription and translation level(P<0.05);the SREBP-2 expression of low doses group had no significant difference at transcription level(P>0.05)and significantly lowered at translation level(P<0.05);in the high and low doses group,the HMGCR expressions were significantly lowered at transcription and translation levels(P<0.05),these results suggested that the Eupolyphaga steleophaga could affect the synthesis of cholesterol,and high dose was more effective.Compared with control group,the expression FXRa of high dose group was significantly lowered at transcription and translation levels(P<0.05),the expression FXRa of low dose was significantly lowerd at transcription level(P<0.05)and no significant difference at translation level(P>0.05);the CYP7A1 expressions were at transcription and translation level in the high and low doses group(P<0.05),and these results indicated that Eupolyphaga steleophaga could promote the synthesis of bile acid in cholesterol balance adjustment.5)At the end of the treatment,the detections of AST,ALT and AST/ALT showed no significant difference between the four groups(P>0.05),indicating that high-dose and low-dose Eupolyphaga steleophaga did not improve liver injury factors.Based on the model of anti-hypercholesterolemia by Eupolyphaga steleophaga,five differcet algorithms were used for evaluating the optimal reference genes in the liver,spleen,kidney and adrenal gland.The optimal reference genes were used for normalizing cholesterol metabolism related genes to explore the pharmacology of Eupolyphaga steleophaga.The study has the following innovations:1)GeNorm,BestKeeper,NormFinder,ACt and RefFinder were applied to calculate the expression stability of nine candidate reference genes in the liver,spleen,kidney and adrenal gland for the model,and the optimal reference genes for four tissue were obtained to improve the accuracy of qPCR results.2)The important genes in cholesterol absorption,outflow,synthesis and transformation pathways,were analyzed for the study at the transcription and translation levels,and revealed the biological principles and mechanisms related to the regulation of sterol metabolism by Eupolyphaga steleophaga.According to the experimental results,the following conclusions are obtained:1)The model of hypercholesterolemia and anti-hypercholesterolemia rabbit can be established by Harbin white rabbits.2)It can improve the accuracy of evaluation results using GeNorm,BestKeeper,NormFinder,?Ct and RefFinder.2)The optimal reference genes in the model were Hprtl and B2m in the adrenal gland,Gapdh and B2m in the liver were,Gapdh and Ywhaz in the spleen,Ppia,B2m and Gapdh in the kidney,which can improve the accuracy of the results of fluorescence quantitative PCR for the analysis of gene expression related to the pharmacological effects of Eupolyphaga steleophaga.3)The anti-hypercholesterolemia mechanism of Eupolyphaga steleophaga may be related to the regulation of the following gene expression:up-regulating the transcription and translation expression of LDLR gene and improving cholesterol absorption;up-regulating the transcription and translation expression of LXR? and ABCA1 genes to promote cholesterol efflux;down-regulating transcription and of SREBP-2 and HMGCR to reduce cholesterol synthesis;down-regulating the transcription and translation expression of FXR?and up-regulating the transcription and translation expression of CYP7A1 for promoting the transformation of cholesterol.4)The current dose of Eupolyphaga steleophaga could not influence the liver function,which could be safely used for hypercholesterolemia treatment. |
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