| BackgroundChronic liver disease is a "global public health problem" which threat to human health and to spend a lot of health resources. Our country is badly suffered of liver disease, the incidence and prevalence of viral hepatitis is very serious. In addition, with the increasing living standards, cirrhosis of the liver caused by a fatty liver and liver fibrosis also grave threats the further development of the national economy and also poses a serious threat to people’s health. Therefore, actively explore the mechanisms of liver fibrosis is not only an important issue for the prevention and treatment of liver fibrosis, but also the protection of our limited medical resources and maintain the sound development of the social economy.Liver fibrosis is a pathological process caused by a variety of pathogenic factors intrahepatic connective tissue dysplasia and intrahepatic excessive deposition of extracellular matrix. Liver fibrosis is an important intermediate links chronic disease of various liver disease to cirrhosis or liver cancer. Activation of hepatic stellate cell is considered to be the core of the development of liver fibrosis, many factors which induce fibrosis are take HSC as the final target cells. Activated HSC get the ability of proliferation and migrated to the entrance of blood vessels of sinusoidal.Contraction of HSC narrowed sinusoidal diameter and increasesde the resistance. Currently, increased intrahepatic vascular resistance is considered to be an important factor in causing portal hypertension, liver microcirculation disturbance is one of the important factors that lead to portal hypertension. Studies have shown that treatment of portal hypertension is the key to controlling sinusoidal endothelial dysfunction and the inhibition of angiogenesis. Thus, inhibition of HSC activation and reduce excessive portal pressure became a key prevention of liver fibrosis.Liver has the system of renin-angiotensin-aldosterone(RAAS).Liver RAAS is closely related to liver fibrosis, RAAS in liver may be involved in the regulation of intrahepatic portal hypertension. Studies have shown that aldosterone promote HSC contraction effect, however, the regulation mechanism of aldosterone on contraction and migration of HSC has few relevant literature. Whether spironolactone, aldosterone receptor antagonist, can reduce intrahepatic resistance, ease of portal hypertension by inhibiting HSC contraction also has few relevant literature. Therefore, this research was to find the control mechanism of aldosterone on HSC contraction and migration and to explore the mechanism of action of spironolactone in the remission of liver fibrosis and the treatment of portal hypertension.HSC can make contraction by calcium-dependent pathways and non-calcium-dependent pathway. Recent studies suggest that calcium-dependent pathways for HSC contraction only play a secondary role. In non-calcium-dependent pathway, RhoA-ROCK pathway may play a dominant effect. Rho family proteins regulating cell contraction and migration by regulating the actin cytoskeleton reorganization, while studies have shown that activation of RhoA and ROCK either by inhibiting NO that sources from endothelial cell, but also by reduce the formation of eNOS expression directly promotes cell contraction and vasoconstriction induced portal hypertension.In recent years, non-genomic regulation of aldosterone taken the attention of researchers. Differ with the regulation of genome, there was no nuclear transfer in non-genomic regulation, so the mineralocorticoid receptoractivation does not take part in the regulation of target gene transcription. Non-genomic effects was fast, efficient features, and important for study of cell contraction, migration, endothelial dysfunction, angiogenesis and other functions. Among the non-genomic regulation, the non-genomic effects mediated by caveolin-1is one of the most important category.Caveolae are flask-shaped structure formed by the membrane retraction, it is rich in cholesterol and sphingolipids. Caveolae can be used as signal molecules temporary anchoring platform, and related to the events of membrane transport, signal transduction, calcium channel, and many other cellular lipid recycling and so on.Caveolin protein is the major structural protein of caveola, cav-1is the most widely distributed and most comprehensived one, so it was most in-depth study. Cav-1protein can be used as a signaling molecule scaffold, the MR, Src, PDGFR, RhoGTPase and some other signaling molecules can be limited in caveola, and the downstream proteins of these molecules can be transferred to caveola, resulting rapid regulatory effect through crosstalk. In caveolae-mediated signaling pathways, src kinases are the key intermediary in non-genomic pathways of cav-1. It can further regulate downstream pathways of PI3K/Akt, NADPH oxidase, Rho GTPase and so on. Notably, Src can promote cav-1Tyr14phosphorylation sites, on the one hand caused the release of eNOS which was inhibited by binding to cav-1and generating NO; on the other hand, src can active Rho GTPase to promote cell contraction and migration. Since the MR can be translocated into the caveola after activated, so the non-genomic effects of aldosterone, such as migration, inflammation, NO synthesis, angiogenesis, and oxidative stress and so on, were most related with caveola.In recent years, the mechanism of oxidative stress response in organ fibrosis acted more and more attention of researchers. Oxidative stress is the process that body or cells excessive production of ROS, the imbalance of the process oxidant/antioxidant and weakened of endogenous antioxidant defenses leads to tissue damage. ROS is mainly produced in the mitochondria and microsomes redox electron transfer process, in addition, the reaction of catalytic process of NADPH oxidase, NO synthase and cyclooxygenase can be associated with ROS generation. Extensive literature confirms that oxidative stress plays a key role in organ fibrosis development process. Almost all of the varieties of risk factors about liver fibrosis make oxidative stress pathways as a part of regulation in hepatic fibrosis:intrahepatic ROS can not only directly stimulate the excess HSC activation, proliferation, migration, promote HSC contraction and induced HSC produce large amounts of extracellular matrix and collagen production, still by inducing liver cells, Kupffer cells generate large libraries of inflammatory mediators and cytokines, such as PDGF, TGF-β-induced activation of HSC in indirect pathway. Thus, oxidative stress for the development of liver fibrosis also plays a key role.In mesangial cells, epithelial cells of the ureter and many other cell types, aldosterone plays a role of inducing ROS generation.There are also studies confirmed the local RAAS was important in promoting oxidative stress in tissues and organ fibrosis. As mentioned above, in the course of liver fibrosis, local RAAS was also actived, synthesis and secretion of aldosterone in HSC itself were significantly higher. Therefore, the exploration of how does aldosterone regulate oxidative stress pathways to induced HSC migration and contraction during the formation of liver fibrosis becomes the focus of our research. As caveola can assemble the mineralocorticoid receptor and NADPH oxidase and some other signal molecule, it was visible that caveola and aldosterone stimulation are closely related to intracellular signal transduction and oxidative stress, so we speculate that aldosterone may be mediated non-genomic dependent induction of HSC through caveolae pathway, and regulated by excessive production of ROS. And thus realize the HSC activation through RhoA/Rock pathway and induces proliferation of HSC contraction, migration and so on.ObjectiveThis paper intends to study the influence of aldosterone to the level of oxidative stress within the HSC via caveolae-mediated non-genomic pathways, and realize the regulate and control of RhoA/Rock-related signaling pathway. In-depth explorate the mechanism about aldosterone regulating the contraction, migration of HSC via cav-1-related non-genomic pathways, to providing new clues to elucidating the formation of portal hypertension in aldosterone regulation mechanisms and provide new targets for prevention and treatment of portal hypertension and liver fibrosis.Methods1, Isolated and cultured primary rat hepatic stellate cells:Combined use the isolated perfused and in situ perfusion, density gradient centrifugation and differential adhesion to isolated and cultured primary rat hepatic stellate cells. Cell purity were identified by immunofluorescence staining and use hepatic stellate cell specific marker protein a-SMA, select the generation of2-5vitro rat hepatic stellate cells in vitro experiments.2, Caveolin protein extraction experiment:Use10cm cell culture dishes culture primary rat hepatic stellate cells, the fusion of the cells within the culture dish to be80%, starved for12hours and give the relevant drug intervention, after the completion of treatment, cells were lysed and homogenized, sucrose density gradient centrifugation and the proteins purified by precipitation with cold acetone, Western blot assay the protein content change.3, The experiment of cholesterol depletion and reloading:Dropping MCD solution to the primary culture of rat hepatic stellate cells, put the cells into incubator and incubated30min, then gently rinse the cells with PBS buffer to depletion of intracellular cholesterol. dissolve lmg cholesterol in5ml MCD with concentration of50nM. The solution added to the cell culture medium after treatment of cholesterol depletion, then put the cells into incubator and incubated30min, cholesterol reloading process was completed.4, Transwell cell migration assay:Making primary rat hepatic stellate cell suspension, counting, adding the serum-free and20%FBS to the upper and lower chambers respectively. After applying the appropriate treatment, the cell were cultured in incubator with appropriate time. Disposable the medium. after washing with PBS, the cells were fixed in methanol. After crystal violet staining, Count the number of migrating cells under the microscope.5, Gel contraction experiments:primary rat hepatic stellate cells were seeded onto collagen lattice which were previously prepared. Separate the collagen lattice and bottom of the culture plate after the cells adherented. Applying the appropriate stimulus and an appropriate incubation time. Take the photograph and calculate the remaining collagen lattice area. Analysis the ontraction of collagen lattice.6, Co-immunoprecipitation experiments:Use10cm cell culture dishes culture primary rat hepatic stellate cells. When the fusion of the cells in the culture dish to be80%, starved the cells for12hours and give the relevant drug intervention. Cells were lysed and made cell homogenates. and then conduct antibody incubation, beads were incubated, centrifugation and high temperatures making denaturation of protein in turn, to obtain the target protein. Western blot experiments to test the changing of the target protein.7, Immunofluorescence staining experiments:Preparation of cells seeded and complete the relevant interventions. Use4%poly-formaldehyde fixed cells, and then make the closure of non-specific antigen, followed by the first antibody and the second antibody incubation and nuclear staining were mounted in fluorescence microscopy/confocal microscope to observe and analyze.8, The establishment of animal models:Select364-week-old male SD rats, weighing120-150g. Rats were randomly divided into six groups and accepted surgical treatment respectively. Rats were fasting and water deprivation within24hours after surgery. Since the third day after surgery, rats were given the appropriate medical treatment according to the packet or not special treatment.9, Immunohistochemistry experiments:Preparing tissue specimens of paraffin, dewaxing, hydration and use high temperature to make antigen retrieval and then inactivating endogenous peroxidase, blocking non-specific antigen, incubating the primary and second antibodies and using DAB to display color. After completion of the color,use hematoxylin to stain nucleus. Then dehydration, transparent, mounted, and take the pohtograph under the microscope.10, Preparate cell or tissue homogenates. Then centrifugal, determinat the concentration and make denaturation of the protein at a high temperature.By the means of electrophoresis to get the target protein. Test the relative expression of the target protein.Result1,10-7M of Aldo can significantly promote the phosphorylation of protein cav-1and protein c-src; when the stimulation time of Aldo was get to30min, the phosphorylation of protein cav-1and protein c-src in HSCs get to the peak.2, The gel contraction experiments shown:gel lattice area was significantly decreased in aldosterone stimulation group, after the intervention by inhibitor, gel lattice contraction was inhibited. At cell migration test, aldosterone significantly increased the migrattion number of cells. After application of inhibitors intervention, the number of migrating cells was significantly reduced compared with aldosterone stimulation group.3, Co-immunoprecipitation experiments suggest:30min after stimulated by aldosterone, interactions between protein MR and c-src protein were significantly enhanced in primary rat hepatic stellate cells, while Aldo stimulation does not induce or change the interactions between protein cav-1and protein MR.4, Cholesterol depletion and reloading experiments suggest:After depletion of cholesterol in the primary rat hepatic stellate cells, the action of activation and phosphorylation of aldosterone on protein cav-1and c-src was inhibited; after cholesterol reloading, the capacity of Aldo on protein cav-1and c-src is restored.5, Intracellular reactive oxygen detection experiments shown:Aldo can significantly promote the primary rat hepatic stellate cells ROS generation, after applying the appropriate inhibitor intervention, the role of Aldo on promoting ROS generation is suppressed.6, Western blot experiments and GST-Pull Down experiments shown:Aldo stimulation significantly increased primary rat hepatic stellate cells expression of protein NOX4and activation of protein RhoA. Aldo can promote the phosphorylation of protein moesin and protein mlc and promote the activation of protein a-SMA and protein collagen. After applied inhibitors block, the responses of these proteins in HSCs to the stimulation of Aldo was significantly weakened. 7, Rats model assessment tips:In BDL groups, the Aldo levels in serum were significantly elevated. In BDL groups’liver tissue, hydrogen peroxide and hydroxyl proline content increased significantly, liver fibrosis were severely. Rats in treatment groups, Aldo, hydrogen peroxide and hydroxyproline content were increased, but significantly lower than the BDL group,the degree of hepatic fibrosis were between the control group and the BDL group.8, Liver tissue immunofluorescence double staining suggest:In BDL rats liver, the intensity of fluorescence behalf protein cav-1and protein a-SMA were significantly enhanced, and the dying position were mainly distributed in sinusoidal portal area and around small blood vessels, brightness range in drug treatment groups were significantly reduced compared with BDL group.9, Liver tissue immunohistochemical double staining Show:NOX4protein in rat liver and a-SMA protein expression was significantly increased in BDL group, and stained cells were distributed in sinusoidal tend Ministry periportal etc, in drug-treated groups, the number of stained cells in the liver tissue was significantly lower than the BDL group.10, Western blot experiments of tissue protein and GST-Pull Down experiments show:In the liver of BDL group, the expression of protein cav-1, protein NOX4, protein collagen and protein a-SMA were significantly increased. At the same time, the activation of protein RhoA, protein moesin and protein mlc were significantly enhanced. the expression or phosphorylation of these proteins in drug-treat groups were inhibited in different levels.Conclusion1, Aldo can activate HSCs and promote the contraction and migration of HSCs by raising the oxidative stress levels in primary rat hepatic stellate cells and increasing the intracellular ROS production. 2, Aldo can activate HSCs and promote the contraction and migration of HSCs by raising the oxidative stress levels in primary rat HSCs via caveolae-mediated non-genomic pathways.3, The high level of Aldo and ROS in BDL rat liver can be reduced by receptor antagonist of Aldo or antioxygen.4, By promoting the expression of caveolin-1and protein NOX4in HSCs, Aldo induced the activation of HSCs and promote the contraction and migration of HSC to sinusoidal blood vessels or periportal in BDL rat liver. |
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