Studies On The Role Of Curcumol-induced HSC Necroptosis In Anti-hepatic Fibrosis And The Underlying Mechanism

ObjectiveHepatic fibrosis caused by multiple chronic liver injuries,is a known contributor to cirrhosis,and even liver cancer.Effective prevention and treatment are still a difficult problem in the medical field.It has been recognized that activation of hepatic stellate cell(HSC)is the core of liver fibrosis.During liver injury,the morphology and function of HSCs are destroyed and HSCs could convert into myofibroblasts(MFB).Activated HSCs produce fibrotic cytokines and growth factors,and sustainedly stimulate damaged liver through paracrine and autocrine,which result in excessive accumulation of extracellular matrix(ECM)in the portal area and hepatic lobules,ultimately inducing liver fibrosis.In addition,activated HSCs migrate and adhere to the sinusoidal endothelium more easily,further stabilizing the basement membrane,impeding the exchange of hepatic cells,and further aggravating liver fibrosis.Therefore,targeting the inhibition of HSC activation and interfering with its biological behaviors such as migration and adhesion,is an effective strategy for the treatment of liver fibrosis.The highly effective and non-toxic natural active ingredients for the treatment of liver fibrosis has become a research hotspot in the field of liver diseases.Based on a large number of previous literature studies,we found that curcuma zedoary is an indispensable part of traditional chinese medicine for liver diseases such as cirrhosis.The volatile oil is an effective component of curcuma zedoary.The curcuma zedoary oil preparation has been collected by the chinese pharmacopoeia as an anti-cancer and anti-viral drug.Curcumol is one of the active ingredients of anti-cancer,anti-viral and anti-bacterial effects of curcuma zedoary volatile oil.It has been reported that curcumol inhibits HSC activation in vitro,but its anti-fibrosis effect needs to be confirmed,and its in-depth mechanism remains to be further revealed.Numerous studies have shown that clearance of activated HSCs by cell death is an important strategy for the treatment of liver fibrosis.Recent studies have shown that necroptosis is involved in liver cancer,fatty liver disease progression,and drug-induced necroptosis could inhibit HSC activation in vitro.Therefore,our research shows that curcumol can improve carbon tetrachloride(CCl4)-induced liver fibrosis by promoting HSC necroptosis.Curcumol-induced necroptosis inhibits the nuclear factor-kappa B(NF-?B)signaling pathway that promotes cell growth,further suppresses periostin(POSTN)-mediated migration and adhesion of activated HSC,and then exerts anti-fibrosis effect.This study aims to clarify the effect of curcumol against hepatic fibrosis and to reveal the underlying mechanism.This result will provide experimental evidence that curcumol is a potential candidate for the treatment of liver fibrosisMethods1.Cell levelThe immortalized human hepatic stellate cell line LX2 was applied for the experiment in vitro Cell Counting Kit-8 was used to determine the curcumol concentration for LX2 cells in vitro.Real-time PCR,Western blot and immunofluorescence staining were performed to detect the expression of target genes and proteins in LX2 cells.The cell death of LX2 cells was measured by trypan blue staining.Transmission electron microscopy was used to observe the characteristics of necroptosis in LX2 cells.The levels of LDH,ATP,MDA,SOD,GSH and POSTN in LX2 cell supernatant were detected by kit.The binding of RIPK1 to RIPK3 in LX2 cell was detected by protein immunoprecipitation.JC-1 staining was used to detect mitochondrial membrane potential in LX2 cell.DCFH-DA and MitoSox Red fluorescent probe were respectively performed to measure reactive oxygen species and mitochondria peroxides in LX2 cell.Cell scratch and transwell chamber are for evaluating cell migration.2.Animal levelA classical murine liver fibrosis model was built through intraperitoneal injection of CCl4,and male ICR mice were treated with low,medium and high doses of curcumol(15,30,60 mg/kg)RIPK3 knock-down mice and POSTN knock-in mice were constructed,and further establish CCl4-induced liver fibrosis model in mice,and then treated with curcumol(30 mg/kg).Hematoxylin and eosin(H&E)staining was used to detect the pathological structure of mice liver.Masson and Sirius Red staining were used to detect collagen deposition in mice liver.The serum levels of liver injury related indicators were detected by commercial assay kits.The inflammation related indicators and liver fibrosis index in serum of mice were detected by ELISA kit.Inflammatory infiltration,HSC activation maker alpha-smooth muscle actin(alpha-SMA)and POSTN in liver of mice were observed by immunohistochemical staining.The expression of fibrosis-related factors in liver of mice was detected by Western blot.Co-localization of alpha-SMA with fibrosis-related factors,necroptosis key factors,and POSTN was detected by double immunofluorescence staining Primary HSC was isolated from each group of mice,and the protein abundance of fibrosis-related factors and necroptosis key factors was detected by Western blot.Results1.Serological and morphological analysis results showed that curcumol significantly reduced CCl4-induced liver injury and inflammatory infiltration in mice.Furthermore,curcumol significantly improved CCl4-induced hepatic steatosis and collagen deposition,and liver pathological angiogenesis in mice,suggesting that curcumol could ameliorate CCl4-induced hepatic fibrosis in mice2.In vitro studies showed that curcumol inhibited the expression of genes and proteins related to HSC activation.Curcumol inhibited HSC activation depending on the occurrence of HSC necroptosis rather than apoptosis.Further experiments showed that curcumol not only increased the expression of RIPK1 and RIPK3 in LX2 cells,but also promoted their interaction,suggesting that RIPK1/RIPK3 complex is necessary for curcumol-induced HSC necroptosis.RIPK3 knockdown exacerbated hepatic fibrosis in CCl4-treated mice,whereas the collagen deposition was improved under curcumol treatment3.In-depth mechanism study found that curcumol induced RIPK1/RIPK3-dependent HSC necroptosis by promoting the production of mitochondrial superoxide and the loss of mitochondrial membrane potential to result in mitochondrial dysfunction in HSC,thereby promoting HSC cell death.In addition,RIPK3 promoted mitochondrial ROS production and depolarization depending on c-Jun NH2-terminal Kinasel/2(JNK1/2)activation.4.Curcumol-induced necroptosis could competitively inhibit the NF-?B signal that promotes cell growth,weakening the secretion and expression of POSTN,further inhibit the migration and adhesion of HSC,and exert anti-fibrosis effectConclusionThis study is the first to prove that curcumol could significantly reduce CCl4-induced liver injury,inflammation and improve liver fibrosis in mice.Mechanistically,curcumol inhibits HSC activation through promoting RIPK1/RIPK3 complex formation,followed by JNK1/2 activation and subsequently mitochondrial dysfunction,which ultimately leads to HSC cell death.Curcumol-induced necroptosis can competitively inhibit the NF-?B signal that promotes cell growth,weakening the secretion and expression of POSTN,further inhibit the migration and adhesion of HSC,and exert anti-fibrosis effect.This study confirms the role of RIPK3-dependent necroptosis in improving hepatic fibrosis induced by curcumol,and provides a powerful experimental basis for the development of anti-hepatic fibrosis drugs targeting HSC necroptosis.