The Effect Of Neuropeptide P On Liver Fibrosis And Activation Of Hepatic Stellate Cells

Liver fibrosis is the final pathological result of various chronic liver diseases.The most important feature of liver fibrosis is an increase in type I and type III collagens and the excessive deposition of extracellular matrix(ECM)in liver parenchyma.Liver fibrosis is a reversible phase during the formation of liver cirrhosis.However,if highly progressive,the fibrotic process eventually leads to severe morbidity and mortality.Currently,early liver fibrosis is a reversible pathological process.However,liver cirrhosis as an end stage of fibrosis is irreversible.Therefore,how to effectively regulate the progress of liver fibrosis,and even inhibit or reversal the process of liver fibrosis is the key point to the treatment of liver fibrosis.A variety of studies showed that activated hepatic stellate cells(HSCs)is the initiating cells and the main source of ECMs in the development of hepatic fibrosis.Several kinds of chronic liver injuries can provoke the activation of quiescent HSCs into a myofibroblast-type phenotype that is proliferative,fibrogenic,and contractile.HSCs are the principal hepatic fibrogenic cells in the production of ECM components,e.g.,collagen,fibronectin and hyaluronic acid,in response to persistent liver injury.The activation of HSCs is a central mechanism underlying liver fibrogenesis.Many signaling pathways and molecules are involved in the regulation of HSC’s activation in liver fibrosis.However,its specific mechanism of action has not yet been folly elucidated.Therefore,it is still necessary to further study the pathways of sustained activation of HSCs in liver fibrosis and to further explore the molecular mechanism of liver fibrosis,which may provide more possible and effective interventions for the prevention and treatment of liver fibrosis in future.The neuropeptide Substance P(SP)is an undecapeptide that belongs to the tachykinin family and is encoded by the TAC1 gene.After SP binds with its receptor NK-1R,it plays an important role in the pathophysiologic processes of pain,hepatitis,myocarditis,cholestasis,inflammation,bacteremia and viral infection through stimulation of immune cell infiltration and cytokine release.Studies have shown that blocking the hepatic afferent nerve or SP receptors can effectively reduce the inflammatory response of liver injury.Moreover,SP can promote hepatocyte protective factors secretion such as interleukin 6(IL-6),interleukin 10(IL-10)and inhibit hepatocyte apoptosis,necrosis and exert hepatocyte protective effect.It is obvious that SP receptor inhibitors can not only inhibit the release of various inflammatory cytokines,but also have anti-apoptotic effects on hepatocytes,SP receptor inhibitors may become the promising drug target for curing liver fibrosis.Our previous study found that SP can promote the expression of NK-1R receptor in liver Kupffer cells(KCs),promote the release of tumor necrosis factor-a(TNF-a),indicating that SP plays an important role in liver injury.However,whether SP is involved in liver fibrosis,especially in the activation and proliferation of HSCs,is largely unknown.Thus,in the present study,we tested the effects of SP/NK-1R on HSC cells activation,proliferation,apoptosis and liver fibrosis.Meanwhile,the underlying mechanism was also investigated.PART I:EFFECT OF SP ON PROLIFERATION,ACTIVATION AND APOPTOSIS OF HSC-T6 CELLSObjectives:The effects of serial concentrations of SP on the proliferation of HSC-T6 cells were observed with different treatment time.At the same time,the effects of SP on the activation of HSC-T6 cells were detected by oil red staining to select the optimum concentration of SP for the following experiments.Moreover,the cells were co-treated with NK-1R receptor inhibitor and the apoptosis rate was also calculated.Western-blot assay was performed to detect the expressions of protein that are associated with the activation of HSC-T6 cells.Methods:1.HSC-T6 cells were cultured in high glucose DMEM containing 10%fetal bovine serum(FBS)at a series of concentrations of 10,50,100,500,1000 and 5000 nM for 6/12 h,respectively.MTT/LDH assays were performed to detect the cell activity.2.The cells were treated with serial concentrations of SP(10,50,100,500,1000 and 5000 nM).The expression of a-SMA/Collagen I was detected by western blot and oil red staining was performed to measure the activation of the cells.3.The effects of SP on HSC-T6 activation were evaluated by co-culture with NK-1R receptor blocker,L732138.MTT/LDH assays,a-SMA/Collagen I protein levels and oil red staining were all performed.4.Hoechst 33342/P1 double staining and flow cytometry assays were used to detect the apoptosis rate of HSC-T6 cells which were treated with SP and L732138.Results:1.The series concentration of SP(10-5000 nM)treated HSC-T6 cells for 6 h,MTT/LDH tests showed no significant difference between the groups.However,when treated for 12 h,500/1000 nM SP significantly increased MTT values and decreased LDH release in HSC-T6 cells.2.Oil red staining showed that 500/1000 nM SP could significantly promote the release of lipid droplets in HSC-T6 cells,and western-blot showed that 500/1000 nM SP promote the expression of a-SMA/Collagen I.3.The NK-1R inhibitor L732138 could significantly inhibit the promotive effects of SP on HSC-T6 cell proliferation and inhibit SP-induced the release of lipid droplets.Moreover,L732138 effectively inhibit the the up-regulation of a-SMA/Collagen I protein induced by SP in HSC-T6 cells.4.Apoptosis-related test showed that 500/1000 nM SP can inhibit the apoptosis of HSC-T6 cells,which can be blocked by NK-1R receptor inhibitor L732138.Conclusions:1.Lower concentration(10-100 nM)of SP had no significant effect on the activity of HSC-T6 cells in a short period of time(6 h),HSC-T6 cells treated with 500/1000 nM SP for 12 h significantly promoted cells activity,inhibiting apoptosis and promoting cells activation.2.L732138 can block the promotion of cell activity and activation in HSC-T6 cells induced by SP.L732138 promoted the apoptosis of HSC-T6 cells,indicating that L732138 can partially inhibit the liver fibrosis induced by SP in vitro experiments.Part II:The effects of SP on liver fibrosis in CCl4 induced rat liver fibrosis modelsObjectives:The rat model of hepatic fibrosis induced by CCl4 was established and the rats were treated with the SP receptor NK-1R inhibitor L732138 at the same time.HE/Masson/Sirius red staining and western-blot were used to evaluate the effects of SP and L732138 on liver fibrosis.In addition,the rat serological indicators were measured to assess the effects of SP and L732138 on liver function.Methods:1.The establishment of CCl4 rat model of liver fibrosis:six-week-old adult male wistar rats(body weight:180-220 g)were intraperitoneally injected with 50%CCl4 olive oil(1 ml/kg)every Tuesday and Friday for a total of 6 weeks,twice a week to establish CCl4 rat liver fibrosis models.2.Animal grouping and drug treatment:18 wistar rats were randomly divided into 3 groups with 6 rats in each group,including normal saline control group,CCl4 model group and CCl4 L732138 drug intervention group.The rats in model group were treated as above.Normal saline treatment group:given the same dose of saline olive oil mixture(1:1 mixture).The drug intervention group was intraperitoneally injected with L732138(0.5 mg/kg)on the day prior to CCl4 treatment(every Monday and Thursday)for 6 weeks,twice a week.At 24 h after the last administration,all rats were anesthetized with 10%chloral hydrate,the livers and blood samples were collected quickly.The rat liver tissues were fixed in 4%paraformaldehyde.Some fresh liver tissues were stored at-80 ℃.3.Liver tissue immunohistochemical staining:Determination the expressions of SP and NK-1R in CCl4 model rats.Freshly-harvested liver tissues were embedded in paraffin,sectioned and antigen-fixed.Then the tissues were blocked with 1%BSA.The primary anti-SP/NK-1R antibodies were used to treated with the tissues overnight in cold room.The fluorescent secondary antibodies were stained for 2 h at room temperature.Then observation and take pictures with the florescent microscope.4.Histological staining:take 4%paraformaldehyde fixed liver tissue and embedded in paraffin.Cut the tissues for 5 μm thick sections and hematoxylin and eosin(HE)staining were performed and the degree of hepatic fibrosis and inflammatory infiltration were observed under light microscope.Masson and Sirius red staining were also performed to further observe the collagen deposition in different groups.5.Western-blot was used to detect the expression of a-SMA/Collagen I in liver fibrosis,and the degree of hepatic fibrosis in each group was evaluated.6.Determination of hydroxyproline content in liver:hydroxyproline content in liver tissue can reflect the degree of liver fibrosis.Take 4%paraformaldehyde fixed liver tissue,homogenizer grinding,6N hydrochloric acid hydrolysis overnight,10N NaOH neutralized,respectively.After treated with citric acid,chloramine T and perchloric acid were added,and finally heated at 65 ℃ for 20 min,microplate reader measured absorbance value at 570 nm.According to the standard hydroxyproline standard curve,the content of hydroxyproline in each sample were calculated.7.Serum biochemistry:Determination of serum ALT/AST levels in rats.Fresh plasma was taken from each group.After centrifugation,the supernatant was collected.ALT/AST levels in each experimental group were determined according to the instructions of ALT/AST test kit to evaluate the degree of liver injury in each group.Result:1.Immunohistochemical staining results showed that the expression of SP and NK-1R were significantly increased in CCl4 model rats compared to the control group.2.HE staining results showed that there was no significant change in the normal rats.CCl4 model group rats showed a large number of liver vacuoles,hepatocellular necrosis,inflammatory cell infiltration and fibrillation.L732138 can inhibit CCI4 mediated liver fibrosis to a certain extent.Masson/Sirius red staining showed that a large number of fibers were found in the model group,and L732138 could reduce the degree of liver fibrosis compared to the model group.3.The results of western-blot showed that the expression of a-SMA/Collagen I in CCl4 model group was significantly higher than that in the normal control group;L732138 significantly reduced the expression of a-SMA/Collagen I level,but still higher than the normal control group.4.The results of hydroxyproline in liver tissue showed that the concentration of hydroxyproline in the CCl4 model group was significantly higher than that in the normal control group,while the L732138 treatment group was significantly lower than the model group,but still significantly higher than the normal control group.5.Serum biochemical tests showed that ALT/AST was significantly increased in the CCl4 model group,while L732138 treatment could inhibit the increasing of ALT/AST.Conclusion:1.In CCl4 rat liver fibrosis models,both the SP and NK-1R protein levels were significantly increased.2.NK-1R receptor blockers can reduce liver injury to some degree and reduce the degree of liver fibrosis and exert anti-liver fibrosis effects.Part III The mechanism of the SP affect hepatic stellate cell proliferation/activation and liver fibrosisObjectives:Preliminary investigate the mechanism of SP affects the activation of hepatic HSCs and liver fibrosis.Methods:1.To exclude the influence of cell species,the experiments were carried out simultaneously in rat HSC-T6 and human hepatic stellate cell line LX2.After treatment with different drugs,the total protein was extracted and the protein levels of TGFβ1/Smad-3 which was highly associated with liver fibrosis were determined by western blot.2.In order to further verify whether SP can affect the activation of hepatic cells and hepatic fibrosis through the TGFβ1/Smad-3 signaling pathway,we treated HSC cells with TGFβ1 receptor inhibitor SB431542.MTT/LDH assays were performed to detect the cell viability and oil red staining for cell activation.Moreover,Western blot was also used to detect the expression of a-SMA/Collagen I.3.To exclude the influence of single drug on hepatic stellate cell activation and liver fibrosis,we used another NK-1R receptor antagonist RP67580 to treat cells.MTT/LDH was used to detect cell proliferation and western-blot to detect the effect on liver fibrosis related proteins α-SMA and Collagen I.4.The endoplasmic reticulum stress(ERS)associated protein Caspase-12 was also measured in CCl4 rat liver fibrosis models.Results:1.500/1000 nM SP can significantly promote the expression of TGF-β1 and p-Smad-3 in HSC-T6 cells and LX2 cells;NK-1R receptor inhibitor L732138 can obviously inhibit the up-regulation of TGFβ1/Smad-3 signaling pathway induced by SP.2.The results of oil red staining showed that the TGFβ1 inhibitor SB431542 significantly inhibited the release of lipid droplets in HSC-T6 cells and inhibited the activation of HSC compared with 1000nM SP group.The results of MTT/LDH assays showed that SB431542 could inhibit the proliferation of HSC-T6 cells and reduce the expression of a-SMA/Collagen I.However,compared to the L732138 treatment group,the difference was statistically significant,indicating that the mechanism by which L732138 inhibits cell proliferation/activation and hepatic fibrosis does not completely via the TGFβ1/Smad-3 signaling pathway.3.Similar to the effects of L732138 on hepatic stellate cell proliferation and activation,another NK-1R receptor blocker RP67580 also inhibited the promotion of hepatic stellate cell proliferation and activation induced by SP,and inhibited the expressions of a-SMA/Collagen I.4.L732138 inhibited the expression of Caspase-12 in the CCl4 induced fibrosis livers,indicating that L732138 could inhibit liver fibrosis by inhibiting the endoplasmic reticulum stress in the liver,not only by TGFβ1/Smad-3 signaling pathway.Conclusion:1.SP promotes hepatic stellate cell proliferation/activation and liver fibrosis through TGFβ1/Smad-3 signaling pathway.2.The mechanism of L732138 inhibiting cell proliferation/activation and hepatic fibrosis did not completely through the TGFβ1/Smad-3 signaling pathway.3.L732138 can inhibit liver fibrosis by down-regulation the endoplasmic reticulum stress in the liver.