Inhibitory Effects Of Novel Chinese Herbal Formulation Via Regulation Of Bile Acid Enterohepatic Circulation And Involved Mechanisms

Background and ObjectiveAtherosclerosis,of which the morbidity and mortality is on the top of diseases spectrum,seriously does harm to global health.During recent years,the incidence of such disease especially increased rapidly in China,leading to great economic and society burden on nowadays China.Hypercholesterolemia was proved to be the key of atherosclerotic lesions' progression.Statins,PCSK9 inhibitors and other drugs had been shown to significantly decrease blood cholesterol levels,which would reduce the incidence of relative cardiovascular diseses.Bile acids were confirmed to play a privotal role in lipid metabolism,which could promote the intestine absorption of fat and fat-soluble vitamins.Only around 5% of the secondary bile acid in the intestine was shown to be excreted in the form of stool,which would be the most important way to eliminate cholesterol from the body.Hyperlipidemia and other AS pathogenic factors were thought to be classified as "blood phlegm" within the traditional Chinese Medicine theory,and while the abnormal levels of intestinal lipid and bile acid would be defined as "intestine phlegm",both of which were shown to participate in the initiation and progression of AS.Based on the above,the anti-atheroslerosis effects and involved mechnisms of the novel Chinese herbal formulation derived from classical prescriptions were investigated using atherosclerotic animal models causing by combination of hyperlipidemia diet and femoral artery injury.Methods1.Effects of Chinese herbal formulation on serum blood lipid and AS lesions in AS model mice:1)AS models were established by combination of hyperlipidemia diet and femoral artery injury in mice.Such mice were then randomized into control,Chinese herbal formulation and atorvastatin as positive control groups.Plasma cholesterol and triglyceride levels were evaluation after 12-week high fat diet,and the differences of HMGR,NPC1L1 and SR-B1 on liver and other relative tissues were investigated by quantitative PCR;2)Plaque burden were investigated by oil red O staining of aortic root and relative parameters including intima-media thickness ratio and others were quantified using Image J software.2.Protective effects of Chinese herbal formulation on intestinal barrier function and meanwhile decreasing the systematic inflammation stress on mice: 1)intestinal permeability was explored by determine the plasma FITC-levels among mice from different groups,which were done 10 minutes after injection of fluorescent dye into the intestinal lumen.And the proliferative rates of intestinal epithelial cells were examined using Ki-67 staining.In addition to the above,expressions of mucin and intestinal barrier tight junction related gene were analyzed;2)Plasma IL-1 beta,IL-17 A,IFN-,IFN-,GM-CSF levels of mice were examined using commercial CBA kits by flow cytometry.3.Effects of Chinese herbal formulation on the bile acid's enterohepatic circulation and involved mechanisms: 1)Expressions of hepatic bile acid synthesis and transport related genes of mice from different groups were determined to explore the potential effects on bile acid enterohepatic circulation;2)UPLC-MS/MS was used to evaluate the difference of cholic acid,deoxycholic acid and others in the plasma and faeces.Result1.Effects of Chinese herbal formulation on decreasing serum cholesterol and inhibiting atherosclerotic lesions in AS mice: After 12 weeks of treatment,total plasma cholesterol levels,aortic plaque burden and intima-media thickness ratio among mice given by Chinese herbal formulation significantly decreased,compared with those of control group(p < 0.05).But gut NPC1LI1 and SR-B1 gene expressions among such mice showed significantly increased(p < 0.05),with no significant effect on plasma triglyceride level and the expression of hepatic HMGR and ABCG5/8(p>0.05),which were different from the response of mice receiving statins treatment.2.Protective effects on intestinal barrier function: After 12 weeks of treatment,the intestinal permeability among mice given with Chinese herbal formulation were significantly decreased,compared with those among mice fed with fat diet only(p < 0.05).Behind mechanism might be associated with a significant increase of intestinal MUC1,Cldn1,Ocln,IL22 R alpha 1 gene expression(p < 0.05),and a significant reduction of intestinal IL-10R? expression(p < 0.05).Other genes of the intestinal barrier function,such as MUC3,Cldn2 and Tjp did not show great changes among mice given of Chinese herbal formulation(p>0.05),suggesting the potential selective protection on intestinal barrier function.Chinese herbal formulation can significantly decrease the levels of pro-inflammatory cytokines IL-6,IL-12,IFN-,MCP-1(p < 0.05),but significantly increase the level of anti-inflammatory factor IFN-?(p < 0.05).3.Effects on regulating enterohepatic circulation of bile acid: After 12 weeks of treatment,plasma levels of TCDCA,TCA,TDCA,TLCA and fecal DCA levels among mice given of Chinese herbal formulation significantly increased,compared with those of mice fed with fat diet only(p < 0.05),but fecal levels of CDCA and TCDCA and plasma DCA levels significantly decreased(p< 0.05).And meanwhile expressions of hepatic BSEP,Oatp2 gene and intestinal Ost alpha / beta,FXR gene among mice given with Chinese herbal formulation group were showed to significantly increase(p < 0.05),while expressions of intestinal ASBT,IBABP,FXR,FGF15 gene and hepatic FGFR4 gene were proved to significantly decrease(p < 0.05),which implied that such Chinese herbal formulation could exert effects on enterohepatic circulation pathway of bile acid.In the meanwhile,no significant effects on liver Cyp7a1,Cyp8b1,Ntcp,Oatp1,SHP gene expressions in the mice of TCM group(p>0.05),suggesting the difference with statins on regulation of enterohepatic circulation of bile acid.ConclusionData from our studies showed that Chinese herbal formulation could regulate enterohepatic circulation of bile acid and improve intestinal permeability,which would contribute to the effect of anti AS.Results of our study were reviewed as following: 1)After 12 weeks of treatment,Chinese herbal formulation can significantly decrease plasma cholesterol levels and plaque burden in AS mice.But the protective effects of Chinese herbal formulation were different from statins on hepatic cholesterol synthesis enzyme HMGR expression;2)TCM could significantly reduce the mice intestinal permeability mainly through the up-regulation expressions of MUC1,CLDN1,OCLN genes and decreasing expression of IL-22 related receptors.Meanwhile,it could increase anti-inflammatory factor IFN-? level and inhibit the increase of pro-inflammatory factors IL-6,IL-12,IFN-,MCP-1;3)No significant effects on the expression of bile acid synthesis related genes by Chinese herbal formulation,which suggested a regulatory role on enterohepatic circulation related genes,including the increase of expression of BSEP and Ost?gene and inhibition the expression of ASBT and IBABP genes.And furthermore,TCM could also participate in the regulation of FXR gene expression,which might be an important target for regulation of bile acid.All the above will lead to the following conclusion: such TCM could exert a protective effects on plasma lipid and atherosclerosis burden via regulation of enterohepatic circulation of bile acid and improving intestinal barrier function,in the name of removal of " phlegm in intestine",which was different from the statins and could provide the clinic with potential strategies against atherosclerosis.