MTOR Activation Triggers The Endoplasmic Reticulum Stress By Bile Acid In Intrahepatic Cholestasis Of Pregnancy

BackgroundIntrahepatic cholestasis of pregnancy(ICP)is a pregnancy-associated disorder that mainly develops during the second or third trimester of pregnancy.ICP affects 0.2-2%of all pregnant women worldwide,irrespective of ethnicity and geographical differences.The clinical features of ICP include pruritus and elevated serum total bile acids(TBAs)and liver transaminases in the mother.However,the major concern associated with this disease is the increased risk of adverse fetal outcomes,such as fetal hypoxia,preterm delivery,meconium-stained amniotic fluid and even stillbirth.To date,researchers generally believe that ICP may be associated with immune imbalanced,aberrant estrogen metabolism,genetic variations.But unfortunately,the etiology and mechanisms underlying the fetal complications associated with ICP are complex and not fully elucidated.Thus,a further study of pathogenesis of ICP is extremely important for preventing adverse pregnancy outcomes.As the central regulator of maternal-fetal interaction,the placenta plays a crucial role in maintaining fetal health.During the progression of ICP,placental function is severely disrupted by the accumulation of bile acids,especially toxic bile acids such as lithocholic acid(LCA),taurocholic acid(TCA),deoxycholic acid(DCA)and chenodeoxycholic acid(CDCA).High concentrations of bile acids can induce several morphological abnormalities,including an increased number of syncytial knots and the expression of apoptotic markers in the placental tissue of ICP patients.In addition,bile acids also induce placental inflammation in patients with ICPRThe mammalian target of rapamycin(mTOR),an atypical serine/threonine protein kinase,is a member of the phosphatidylinositol kinase-related kinases(PIKKs)family.mTOR regulates cellular and organismal homeostasis by coordinating anabolic and catabolic processes with nutrient,energy,and oxygen availability and growth factor signaling.mTOR exists in two distinct complexes called Complex 1(mTORC1)and Complex 2(mTORC2).mTORCl promotes protein synthesis largely through the phosphorylation of two key effectors,p70S6 Kinase 1(S6K1)and eIF4E Binding Protein(4EBP).mTORC2 controls proliferation and survival primarily by phosphorylating several of the AGC(PKA/PKG/PKC)family of protein kinases such as Akt.Some previous reports showed that mTOR is an important transducer of endoplasmic reticulum(ER)stress,leading to increased vulnerability of cells to apoptotic cell death.The endoplasmic reticulum(ER)serves several important cell functions,including protein folding,maturation and trafficking,as well as cholesterol synthesis.The quality control of newly synthesized proteins by the ER is essential for normal cell function and survival.However,integrity of the ER is often perturbed by accumulation of unfolded or misfolded proteins,leading to the unfolded protein response(UPR).The UPR alleviates ER stress by reducing immature proteins in the ER,whereas severe or prolonged ER stress leads to activation of the pro-apoptotic UPR and consequent cellular death.High concentrations of toxic hydrophobic bile salts within hepatocytes during cholestasis promotes ER stress,contributing to hepatocellular injury.However,the role of ER stress in the pathogenesis of ICP has rarely been reported.In the present study,we aim to investigate the role of mTOR signaling and ER stress by bile acids in ICP.Methods1.In vivo experiments(1)ICP placenta histomorphology observationThis study was performed in 20 cases of pregnant women with ICP in the obstetrical department of Nanfang Hospital of Southern Medical University from December 2012 to September 2013.20 cases the same period in term pregnant women delivered by cesarean section due to social factors as a control group.HE staining was used to detect histological abnormalities of ICP placental trophoblasts.The number of syncytial knots in each group was counted.(2)ImmunohistochemistryThe expression of p-S6(S235/236)and p-Akt(S473)in ICP group and normal pregnancy group were detected by immunohistochemical method.Using SPSS 17.0 statistical software,The measurement data are shown as the mean±SD.Comparison between groups using ANOVA test.P<0.05 was considered statistically significant.2.1n vitro experiments(1)HTR-8/SVneo cells were treated with LCA,TCA and UDCA.(2)HTR-8/SVneo cells were co-treated with LCA and rapamycin or wortmannin.(3)The expression of p-S6K1(T389),p-S6(S235/236),p-Akt(S473),IREla and Bip protein in HTR-8/SVneo cells was detected by Western blotting.(4)Cell Counting Kit-8(CCK8)was used to detect the cell viability after treated with different concentrations of LCA.(5)Cell Counting Kit-8(CCK8)was used to detect the cell viability after co-treated with rapamycin or wortmannin with different concentrations of LCA.Results(1)Human ICP placenta histological changesCompared to normal placentas,we observe increasing syncytial knots,cytotrophoblast hyperplasia,villi interstitial edema with formation of vacuoles and villous cellulose-like necrosis from ICP placentas.(2)Immunohistochemical study of mTOR signaling pathway in human ICP placentap-S6(S235/236),p-Akt(S473)is mainly expressed in the cytoplasm of placental trophoblast cells.Compare with control group,p-S6(S235/236)and p-Akt(S473)in ICP group increased significantly.(3)Treatment with LCA,TCA and UDCA,the protein levels of p-S6K1(T389),p-S6(S235/236)and p-Akt(S473)in HTR-8/SVneo cells were detected by western blottingThe levels of p-S6K1(T389)and p-S6(S235/236)increased sharply compared with control levels in response to different doses(10,15,20,30,and 40 ?M)of LCA at 1.5h,as detected by western blotting,and a similar pattern was observed for p-Akt(S473).Treatment with LCA at 20 ?M for different times(0.5,1,1.5,2,and 4h)also upregulated p-S6K1(T389),p-S6(S235/236)and p-Akt(S473).TCA and UDCA had no effect on the levels of p-S6(S235/236)and p-Akt(S473)at different doses and times.The three types of bile acids had no profound impact on the expression of S6,Akt,or Actin.(4)Co-treatment with LCA and rapamycin or wortmannin,the protein level of p-S6(S235/236)and p-Akt(S473)in HTR-8/SVneo cells was detected by Western blottingTreatment with rapamycin followed by 15,20,or 30 ?M LCA administration markedly decreased the levels of p-S6(S235/236)to a level similar to the control group,whereas the levels of p-Akt(S473)increased compared with the control group under the same treatment.However,treatment with wortmannin followed by different doses of LCA prevented the increase in the levels of p-S6(S235/236)and p-Akt(S473).The expression of S6,Akt,and Actin was not affected by LCA,rapamycin,or wortmannin.(5)Treatment with LCA,the protein levels of IRE1? and Bip in HTR-8/SVneo cells were detected by western blottingThe levels of ER stress markers including IRE la and BiP increased sharply compared with control in response to treatment with LCA at 20 ?M for different times(1,3,and 6h)in HTR-8/SVneo cells.(6)Co-treatment with LCA and rapamycin or wortmannin,the protein level of IRE la and Bip in HTR-8/SVneo cells was detected by Western blottingTreatment with 20?M LCA followed by rapamycin or wortmannin for different times(1,3,and 6h),we observed that followed by rapamycin treatment,the levels of BiP decreased sharply especially at 3h or 6h,however,rapamycin had no profound impact on the levels of IRE1?.Followed by wortmannin treatment,their levels decreased significantly at 1h,3h or 6h.the expression of IRE la and Bip decreased sharply especially at 2h or 3h.(7)CCK8 test cell survival rate after treated with LCAThe cell survival rate decreased strikingly in response to treatment with 15,20,or 30 ?M LCA in HTR-8/SVneo cells compared with the control group,whereas 5 and 10 ?M LCA had no significant effect on cell survival.(8)CCK8 test cell survival rate after co-treated with LCA and rapamycin or wortmannin15,20 or 30?M LCA treatment caused a sharp decrease in cell survival,whereas rapamycin or wortmannin administration is able to alleviate cell death modestly in HTR-8/SVneo cells.Conclusion1.Endoplasmic reticulum stress triggered by mTOR signaling pathway maybe associated with the pathophysiology of ICP.2.LCA but not TCA or UDCA activated mTOR signaling in vitro.3.Activation of mTOR resulted in induction of ER stress,leading to HTR-8/SVneo cells death.