Study On The Mechanism Of Curcumin In Preventing Cholesterol Stone Formation In Gallbladder

Objective: In the formation of cholelithiasis,especially in the formation of gallstones,the physical and chemical homeostasis of bile duct and bile in gallbladder plays an important role.For the treatment of gallstones,laparoscopic cholecystectomy is currently recognized as the gold standard at home and abroad.Although compared with the traditional open surgery,the damage of laparoscopic cholecystectomy to patients has been reduced to a very low level,but there is still the possibility of serious complications such as biliary tract injury,especially in the center of laparoscopic surgery.Once the injury is formed,it not only increases the treatment time of patients,increases the pain and economic burden of patients,but also intensifies the focus of doctor-patient disputes.Considering that laparoscopic cholecystectomy is a standard treatment,the severity of its complications deserves special attention,and biliary tract injury is also a common cause of medical litigation.Therefore,a more thorough understanding of the pathogenesis of gallbladder cholesterol stone disease,to find effective,non-invasive prevention and treatment of gallbladder cholesterol stone disease method is more and more necessary.Studies have shown that the availability of dietary and bile derived cholesterol in the intestinal cavity and the absorption efficiency of intestinal cholesterol are closely related to the stone-forming status of the population susceptible to calculi.Ezetimibe(EZET),widely used in clinic,can prevent the formation of cholesterol stones in gallbladder by effectively inhibiting the absorption of intestinal cholesterol.However,in clinical practice,EZET alone is not an ideal lipid-lowering agent,so it must be combined with statins to achieve a satisfactory effect.Therefore,we are going to find a better solution from other regulatory factors.Curcumin,a drug that lowers serum cholesterol and may have the same effect as EZET in preventing gallbladder cholesterol stone formation,is coming into focus.Curcumin and its derivatives are phenolic compounds found in ginger and araceae plants,which have the functions of inducing apoptosis,anti-oxidation,anti-inflammation,treating cancer,cholagogic and lowering blood lipid.Curcumin has been shown to lower plasma total cholesterol levels and prevent diet-induced hypercholesterolemia in diabetic patients.Curcumin can also reduce the absorption of exogenous cholesterol by caco-2cells.This effect may be achieved by Sterol regulatory element binding protein 2(SREBP-2)inhibiting the expression of Niemann-pick c1-like 1(NPC1L1)in intestinal epithelial cells.In order to investigate whether curcumin may reduce the formation of cholesterol stones in the gallbladder by inhibiting the absorption of intestinal cholesterol,this study was intended to test gallbladder stone formation in mice fed a high-fat diet through curcumin intervention.Meanwhile,the expression of NPC1L1 and SREBP-2 in intestinal epithelial cells was detected.Studies have shown that curcumin can down-regulate the expression of SREBP-2 m RNA by inhibiting the expression of specific protein-1(SP-1).The SREBP-2 within the cells play a role of transcription factors,need to first via the SREBP divided activated protein(SCAP)will SREBP-2 proteins in the endoplasmic reticulum to the Golgi body,and then by Site 1 Protease(S1P)and Site 2 Protease(S2P)to two-step orderly protease precursor SREBP-2 in Golgi body.The generated mature SREBP-2 can be acquired.The above results are likely to play a role in curcumin’s inhibition of cholesterol absorption in intestinal epithelial cells,and will also be our research direction.Methods: Firstly,an animal model of cholecystolithiasis formation induced by high-fat diet in mice was established.After curcumin intervention,stone formation of cholecystolithiasis in mice was detected,changes in cholesterol and triglyceride in serum and bile of gallbladder were detected,and changes in lipid composition of gallbladder bile in mice were evaluated.Pathological examination of liver and gallbladder tissue changes in mice was done.Expression changes of NPC1L1 and SREBP-2 in mouse intestinal epithelial cells were detected by Western Blot and PCR.Caco-2 cells were used as the research platform to explore the molecular mechanism of curcumin inhibiting cholesterol transport in intestinal epithelial cells.The m RNA and protein expressions of SREBP-2,SCAP,SP-1,S1 P and S2 P were detected after the intervention of curcumin at different concentrations and curcumin at the same concentration at different times in Caco-2 cells.After the intervention of curcumin in Caco-2 cells,the changes of the intracellular distribution of SREBP-2 precursor protein and mature protein were detected by Western Blot through cell distribution extraction.After the intervention of different concentrations of curcumin in Caco-2 cells,the changes of the intracellular distribution of SREBP-2 precursor protein and mature protein were detected by immunofluorescence.Results: Curcumin could inhibit the formation of cholesterol stones in gallbladder induced by high fat diet,and the stone inhibition rate was positively correlated with the dose of curcumin.Piperine can increase the effect of curcumin.Piperine had a significant protective effect on the livers of mice fed a high-fat diet,inhibiting fatty degeneration in the livers,independent of the presence of curcumin.Curcumin reduced liver weight in mice,and immunohistochemistry of the liver and gallbladder showed that curcumin reduced hepatic steatosis and gallbladder wall proliferation.Biochemical tests of serum and bile indicated that curcumin could reduce the content of cholesterol in serum and bile and change the lipid composition of bile in gallbladder of mice.Western Blot and PCR of intestinal epithelial cells showed that curcumin could reduce m RNA and protein expression of NPC1L1,m RNA expression of SREBP-2 and protein content of SREBP-2in mature intestinal epithelial cells.After curcumin intervention in Caco-2 cells,the detection results suggested that curcumin could down-regulate the m RNA expression of SREBP-2,SP-1 and SCAP,but not the protein expression of the precursors SREBP-2and SCAP.The inhibitory effect of curcumin on SP-1 protein expression was short-lived.Curcumin can inhibit the protein hydrolysis process of SREBP-2,reduce the generation of mature srebp-2,and change the distribution state of SREBP-2 in cells.Curcumin decreased the m RNA and protein expression of S1 P,but did not affect the m RNA and protein expression of S2 P.Conclusion: Curcumin can reduce the formation rate of cholesterol stones in gallbladder of mice fed a high-fat diet.With the increase of curcumin dose,the stone formation rate decreased gradually.Piperine can improve the bioavailability of curcumin.Piperine itself did not inhibit cholesterol stone formation in gallbladder of mice fed a high-fat diet,but piperine inhibited fatty degeneration in liver,which was not only superior to curcumin,but also unrelated to it.Curcumin decreased the m RNA expression of NPC1L1 and SREBP-2 in the epithelial cells of small intestine of mice fed a high-fat diet,and the inhibitory effect was dose dependent.Curcumin can reduce the expression of NPC1L1 protein.Curcumin did not affect SCAP m RNA and protein expression.Curcumin inhibited SP-1 m RNA and protein expression,but the effect was short(< 48h).Curcumin can affect the distribution of SREBP-2 in cells,inhibit the proteolytic process of SREBP-2,and reduce the amount of m SREBP-2 protein.Curcumin can inhibit the protein expression of S1 P and further inhibit the protein hydrolysis process of SREBP-2.Therefore,curcumin inhibits the expression of NPC1L1 in small intestinal epithelial cells,possibly by reducing the expression of S1 P,leading to the reduced effect of SREBP-2 in mature cells.