| Background and ObjectiveAtherosclerosis(AS)is the major cause of acute cardiovascular and cerebrovascular events,the main pathological feature of which is lipid-rich plaques.However,no effective strategy to quickly stabilize and induce the regression of the plaques is available.According to traditional Chinese medicine theory,atherosclerosis can be classified as a syndrome of phlegm and blood stasis.Phlegm would obstruct vessels,and as time goes by the nutrient-blood is in stasis.As a result,the phlegm and blood stasis clump together.Data from our previous studies had showed that“eliminating sputum and removing stasis formulation”could inhibit the development of plaques,the involved mechanism of which might be different from statins.Increasing evidences had demonstrated that enterohepatic circulation of bile acid could play a key role in the metabolism of cholesterol,which would be a crucial precursor of cholesterol.Hence,the abnormal reabsorption of bile acids from intestine could be regarded as“sputum in internal organs”.Preliminary in vivo studies have revealed that the fecal levels of bile acids were increased after being given of such drugs,and furthermore the structures of terminal ileum mucosa among mice treated with such drugs were intact.Our study aimed to explore the involved mechanism about how“eliminating sputum and removing stasis formulation”inhibits atherosclerosis by removal of“sputum in internal organs”,which would provide new strategies and methods for future clinic.Methods1.Establishment and validation of human colon adenocarcinoma Caco-2 cell monolayer model:Maturation of Caco-2 cells was evaluated by detecting transepithelial electrical resistance and ALP activity serially,which were done during Caco-2 cells being incubated for 21 days in Transwell insert.Such cells were divided into three groups randomly:control,stimulating with different bile acids and TCM pre-intervention plus challenged with bile acids groups.2.Effects of eliminating sputum and removing stasis formulation on bile acid absorption in Caco-2 cell line and involved mechanisms:1)RP-HPLC with pre-column derivatization was utilized to evaluate the difference of the concentrations of bile acids in the lower compartments of Transwell;2)mRNA and protein expressions of bile acid transport-related genes among different groups were detected to explore the potential mechanism on bile acid absorption by qRT-PCR and Western Blot.3.Effects of eliminating sputum and removing stasis formulation on barrier function and oxidative stress on such cell model:1)Differences of cell monolayer permeability were determined by measuring the concentrations of FITC in the lower compartment and TEER among different groups.Meanwhile,mRNA and protein expressions of tight junction related genes were investigated;2)Generation of intracellular ROS and the ratio of cell apoptosis induced by bile acids were assessed by fluorescence probe and flow cytometry.Results1.Evaluation on Caco-2 cell monolayer model:9 days after cells being incubated in the Transwell insert,TEER had reached to(510.93±44.54)Ω·cm~2,which was higher than the standard of model establishment and significantly increased after 19 and 21 days.8days after cells being incubated,apical ALP activity was higher than basolateral side remarkably(P<0.05).Ratio of the apical versus basolateral ALP activity stood at 2.68:1and 3.44:1 respectively after 19 and 21 days.2.Effects on inhibiting bile acid absorption:1)TCM remarkably decreased the concentrations of CA,GCA and TCA in the lower compartments of Transwell inserts compared with those challenged with corresponding bile acids(P<0.05),but no significant effect was found on the concentration of DCA(P>0.05);2)mRNA and protein expressions of ASBT and OSTβwere increased by CA,GCA and TCA,which were significantly inhibited by TCM(P<0.05),but it had no effect on IBABP mRNA expression(P>0.05).TCM didn’t exert significant effects on the mRNA and protein expressions of ASBT and OSTβchanged by DCA(P>0.05),but it increased mRNA expression of IBABP(P<0.05).TCM inhibited the increased mRNA expression of OSTαstimulated by GCA and TCA(P<0.01),but no similar effects were shown on groups challenged with CA and DCA(P>0.05).In conclusion,TCM could inhibit bile acid absorption in Caco-2 cells effectively,but the effects and involved mechanism differed among different bile acids.3.Protective effects on barrier function of intestinal epithelial cells:1)TCM was shown to keep the cells in low permeable status,increase TEER and mRNA and protein expressions of Ocln and ZO2 remarkably in cells challenged with CA,DCA and GCA(P<0.05).However,no significant effects were uncovered against the pathological effects of TCA(P>0.05);2)TCM was found to reverse the decreased mRNA expression of Cldn1induced by GCA and TCA(P<0.01),but no similar effects were shown against CA and DCA(P>0.05).TCM could up-regulate the Caco-2 cells’ZO1 mRNA expression challenged by DCA,GCA and TCA(P<0.01),but no effects were found against CA(P>0.05);3)Meanwhile,generation of ROS and increased levels of apoptotic cell caused by CA,DCA and GCA were dramatically decreased by pre-incubating the cells with TCM(P<0.05).No significant effects were observed on TCA(P>0.05).In conclusion,TCM was confirmed to exert protective effects on intestinal mucosa,but such effects and behind mechanisms differed among different kinds of bile acids.ConclusionData from our studies showed that“eliminating sputum and removing stasis formulation”could inhibit bile acid absorption and improve barrier function of intestinal epithelial cells.All the above data could lead to the following facts:1)TCM could inhibit the absorption of bile acids by intestinal epithelial cells,which might be associated with decreased mRNA and protein expressions of ASBT and OSTβ;2)TCM could significantly attenuate intestinal barrier dysfunction induced by different BAs via regulation of tight junctions,suppression of oxidative stress and decrease of cell apoptosis.In conclusion,TCM might exert regulative effects on plasma lipid level and progression of atherosclerotic lesions via inhibition of reabsorption of bile acid and improving barrier function of intestinal epithelial cells,namely removal of“sputum in internal organs”. |
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