Role Of TRPC1 In Promoting Liver Fibrosis And Screening Of The Active Components Of Erzhi Pill Targeting TRPC1 And The Efficacy Of Ligustroflavone

1 BackgroundLiver fibrosis is a physiological and pathological process,which is the prosthetic response to liver injury that commonly occurred during liver repair and healing.It is the intermediate stage of the development of various chronic liver diseases to liver cirrhosis and liver cancer,as well as the crucial aspect affecting the prognosis of chronic liver diseases.However,there is still a lack of ideal antifibrotic drugs.It is very important for the discovery of new pharmacological drugs in the identification of potential therapeutic molecules and pathway-specific targets involved in the occurrence and development of liver fibrosis.Canonical transient receptor potential channels（TRPC）are one kind of important voltage-independent cationic channels located on the cell membrane,which participate in a variety of physiological functions and have selective permeability to calcium ions.TRPC1 is one of the earliest cloned mammalian transient receptor potential channels.Previous studies have found that TRPC1 plays an important role in epithelial mesenchymal transformation（EMT）and its expression is increased in human primary hepatocellular carcinoma（HCC）.However,it is not clear how TRPC1 is involved in the occurrence and development of liver fibrosis.It can provide an effective therapeutic target for liver fibrosis on the study of the molecular mechanism and regulatory pathway of TRPC1-mediated liver fibrosis.It has been widely confirmed that liver fibrosis can be improved by traditional Chinese medicine in basic and clinical research.Erzhi pill is a common medicine with characteristics of tonifying the liver and kidney in traditional Chinese medicine.It comes from Fu Shou Jing Fang（Ming Dynasty）and now is recorded in Chinese Pharmacopoeia（2015 edition,Volume I）,which including Ligustrum lucidum Ait.（picked by the Winter Solstice）and Eclipta prostrate L.（picked by the Summer Solstice）.Now it is widely used in clinical treatment on hepatitis,hyperlipidemia and other clinical diseases with yin deficiency of liver and kidney.It is also confirmed that Erzhi pill can treat liver diseases such as liver injury,liver cirrhosis,liver cancer and so on in modern pharmacological studies.Based on this,this paper adopted the idea of“Keeping the Chinese by the West and Keeping Innovation”to study the pathogenesis of liver fibrosis and identify the drug intervention target TRPC1,and then screened the active components of traditional Chinese medicine for the treatment of liver fibrosis based on TRPC1 and verified its effect.2 AimTo study the role and mechanism of TRPC1 in regulating liver fibrosis and provide a new drug target for liver fibrosis.Guided by the dialectical effect of traditional Chinese medicine on yin deficiency of liver and kidney in the middle and late stage of liver fibrosis,the active components based on the TRPC1 target were screened from Erzhi pill,a kind of traditional Chinese medicine for tonifying liver and kidney,and its pharmacological effects were clarified.3 Methods（1）Studies on the role and mechanism of TRPC1 in liver fibrosisThe mouse model of CCl₄-induced liver fibrosis was established,the expression of TRPC1 andα-SMA in mouse liver fibrosis were detected by immunohistochemistry and Western blot approaches;The localization of TRPC1 and Desmin,α-SMA,F4/80,ALB in HSCs,KCs as well as hepatocytes in mouse liver fibrosis were detected by immunofluorescence.In addition,human liver tissue and liver fibrosis tissue were selected,the expression and localization of TRPC1 were observed by fluorescence double staining method.The mouse model of CCl₄-induced liver fibrosis was established to verify the effect of Trpc1 gene knockout on the liver fibrosis formation.The experimental animals were divided into four groups:wild type（WT）,Trpc1^-/-,WT+CCl₄ and Trpc1^-/-+CCl₄.The mouse model of liver fibrosis was induced by intraperitoneal injection of 20%CCl₄（2 m L/kg）twice a week for 6 weeks.The activities of ALT,AST and Alb in serum were detected by enzyme method to evaluate the liver function.The level of MDA in liver tissue was detected by TBA method,the activity of SOD and GSH-px concentration in liver tissue was detected by enzyme labeling method,which evaluated the level of oxidative stress.The level of cell apoptosis in liver fiber tissue was evaluated by H&E staining and TUNEL method.The degree of liver fibrosis injury was evaluated by Masson and Sirius Red staining as well as Hyp content which calculated by sample alkaline hydrolysis.The expression ofα-SMA and Col1a1 in mouse liver tissue was detected by immunohistochemistry and Western blot method to evaluate the degree of HSCs activation and ECM deposition;Western blot was used to detect the expression of TGF-β₁,TβRⅠ,TβR II,Smad4 and Smad2/3 protein and their phosphorylation in mouse liver tissue.Human hepatic stellate cell line LX-2 cells were activated by TGF-β₁ to construct a human hepatic fibrosis cell model in vitro,and a lentivirus transfected LX-2 cells with Trpc1 gene silencing was constructed.The expression of TRPC1 protein was observed under fluorescence microscope and Western blot method to evaluate the transfection efficiency of sh TRPC1.The expression of Col1a1 and ACTA2（α-SMA）genes and proteins were detected by q PCR and Western blot to evaluate the HSCs activation and ECM deposition.The expressions of TβRⅠ,TβR II,Smad4 and Smad2/3proteins and their phosphorylation were detected by Western blot method after Trpc1 gene silencing was stimulated by TGF-β₁.The expression of PDK2 and AKT1 proteins and their phosphorylation after TGF-β₁ stimulation by Trpc1 gene silencing was detected by western blot.Calcium fluorescence probe Fura-2/AM was used to detect the concentration of Ca²⁺in Control and sh TRPC1 cells treated with TGF-β₁.（2）Virtual screening and experimental verification of effective components based on TRPC1 target in Erzhi pillThe chemical constituents of Erzhi pill were collected by Pub Med,CNKI and TCMSP database,and the absorption,distribution,metabolism,excretion and toxicity（ADMET）properties of the collected components were predicted by admet SAR tool,and the chemical composition database of Erzhi pill was constructed.The homologous three-dimensional structure of TRPC1 protein was obtained by homology modeling on Swiss-Model,and the quality of homologous three-dimensional structure of TRPC1 protein was evaluated by ramachandran plot.The flexible docking operation between TRPC1 three-dimensional protein and candidate dominant components was carried out by Discovery Studio software.The binding between candidate dominant components and TRPC1 three-dimensional structure was evaluated by CDOCKER interaction energy as a preliminary index,and the docking results were analyzed by hydrogen bonding and other interactions.The affinity of TRPC1 protein and compound was determined by microthermal electrophoresis（MST）,and its effect on TRPC1-mediated store-operated Ca²⁺entry（SOCE）was verified.The liver fibrosis model of LX-2 cells was established.The effects of LF on the expression of Trpc1m RNA and protein were determined by q PCR and Western blot.（3）Studies on the role and mechanism of LF in CCl₄-induced liver fibrosisThe male C57BL/6J mice were randomly divided into 5 groups.Control group,LF group（20 mg/kg,i.p.,3 times each week）,CCl₄ group,CCl₄+L-LF group（5 mg/kg,i.p.,3times each week）and CCl₄+H-LF group（20 mg/kg,i.p.,3 times each week）.The mouse model of liver fibrosis was induced by intraperitoneal injection of 20%CCl₄（2 ml/kg）twice a week for 6 weeks.From the date of modeling,the drug was given for 6 weeks,3 times a week and every other day.24 hours after the last intraperitoneal injection of LF,serum enzyme method was used to detect the activities of ALT,AST and Alb to evaluate the liver function,TBA method was used to detect MDA level in liver tissue,enzyme labeling method was used to detect the activity of SOD,and GSH-px concentration in liver tissue,which was used to evaluate the level of oxidative stress.Hyp,Sirius Red and Masson staining were used to evaluate the degree of liver injury and liver fibrosis.The expression ofα-SMA and Col1a1 protein in mouse tissues was detected by immunohistochemical staining and Western blot method to evaluate the HSCs activation and ECM deposition.Human hepatic stellate cell line LX-2 was cultured in vitro to set up 4 groups:Control group,LF group（25μM）,TGF-β₁ group,TGF-β₁+LF group（25μM）,immunofluorescence staining and Western blot were used to detect the expression ofα-SMA and Col1a1 protein.The expression and activation of related proteins in TGF-β/Smad pathway were detected by Western blot.4 Results（1）Trpc1 gene knockout or silence inhibited the occurrence and development of liver fibrosisThe results of immunohistochemistry and Western blot showed that the positive staining area and protein expression of TRPC1 increased by 3.4-fold and 2.9-fold,respectively,in CCl₄-induced liver fibrosis mouse model.Cell co-localization study showed that TRPC1 was expressed in hepatocytes,KCs and HSCs;resting HSCs expressed low-level TRPC1 in normal mouse liver,but activated HSCs expressed high level of TRPC1 in CCl₄-induced liver fibrosis mouse tissues.It was further confirmed that the expression of TRPC1 was increased in human liver fibrosis tissues,and was highly expressed in activated HSCs in liver fibrosis tissues.The CCl₄-induced liver fibrosis model of Trpc1^-/-mice was established.The results showed that after Trpc1 gene knockout,AST activity decreased by70%,the ALT activity decreased by 89%and the activity of Alb increased by 1.1-fold in serum,compared to CCl₄group;in liver tissue,the level of MDA decreased by 40%,the content of Hyp decreased by 37%,the concentration of GSH-Px increased by 22%and the activity of SOD increased by 41%.It was indicated that Trpc1 gene knockout could improve liver function and oxidative stress in CCl₄-induced liver fibrosis.The results of HE and TUNEL staining showed that the degeneration,necrosis and inflammatory infiltration of hepatocytes in Trpc1 knockout mice were significantly decreased,and the number of apoptotic cells in CCl₄+Trpc1^-/-group was 2.1-fold lower than that in CCl₄ group.The immunohistochemical staining of F4/80 and TGF-β₁ showed that,compared to the CCl₄group,the infiltration of KCs in Trpc1^-/-+CCl₄ group was significantly lower.The results of Sirius Red staining and Masson staining showed that after administration of CCl₄,the hepatic sinusoidal and Disse’s space fibrosis,portal area fibrosis and bridging fibrosis in Trpc1 knockout mice were significantly less than those in CCl₄ group,suggesting that Trpc1gene knockout can significantly improve CCl₄-induced hepatic fibrosis in mice.Immunohistochemical staining showed that compared with CCl₄ group,the expression ofα-SMA and Col1a1 protein decreased significantly in central vein,hepatic sinusoid space and portal area in CCl₄+Trpc1^-/-group.Western blot results also showed that the expression ofα-SMA and Col1a1 protein decreased by 94%and 41%,respectively,suggesting that Trpc1 gene knockout can significantly inhibit the process of liver fibrosis.Western blot results showed that Trpc1 gene knockout significantly decreased the expression of TGF-β₁,TβRⅠ,TβRⅡ,Smad4,p-Smad2/3 in CCl₄-induced liver fibrosis.Human hepatic stellate cell LX-2 was applied in our study,the human hepatic fibrosis cell model was constructed by TGF-β₁（5 ng/m L）stimulated.The results showed that compared to Control group,the expression of Trpc1 m RNA and protein reached the maximum at 24 h after TGF-β₁stimulation,which was the best time for LX-2 cells activation induced by TGF-β₁.Combined with the results of the first part of the study,it is suggested that TRPC1 may be involved in regulating the activation and proliferation of HSCs and affecting the process of liver fibrosis.After sh TRPC1 transfection into LX-2 cells mediated by lentivirus,after puromycin screening,compared to Control group,transfection of LPP-HSH061231-LVRU6GP-c-200（sh TRPC1）lentivirus could reduce the expression of TRPC1 protein by81%,suggesting that the efficiency of Trpc1 silencing transfection was the best for follow-up experiments.QPCR and Western blot methods showed that after Trpc1 gene silencing induced by TGF-β₁,ACTA2,Col1a1m RNA,α-SMA and Col1a1 proteins decreased by 1.1-fold,1.3-fold,34%,58%respectively compared to induce by TGF-β₁ alone,which was suggested that the specific down-regulation of TRPC1 expression inhibits HSCs activation and ECM deposition to inhibit fibrous formation.Further Western blot assay showed that the protein expressions of TβRⅠ,TβRII,p-Smad2,p-Smad3,Smad4,PDK2 and p-AKT1 in LX-2 cells induced by TGF-β₁ were significantly decreased after Trpc1 gene silencing.It is suggested that the specific down-regulation of TRPC1 expression in HSCs mediates the intracellular signal transduction of TGF-β₁ from the receptor to the nucleus,thus inhibiting the activation of HSCs.sh TRPC1 can also reduce liver fibrosis by inhibiting AKT signal pathway.It was significantly increased Ca²⁺influx and SOCE was activated in LX-2pretreated with TGF-β₁ for 24h.It was significantly reduced Ca²⁺influx and inhibited activation of SOCE by Trpc1 gene silencing pretreated by TGF-β₁.（2）LF could inhibit the activity of TRPC1 in Erzhi pillThrough literature collection and analysis,the compounds without CAS number and Pub Chem CID were excluded,and the properties of ADMET were predicted.Finally,22compounds with good intestinal absorption,no Ames toxicity and no carcinogenicity were selected as the dominant components of ADMET.The homologous three-dimensional structure of TRPC1 protein was obtained by homologous modeling on Swiss-Model.Discovery Studio software was used for molecular docking,and the highest score was Ligustroflavone（LF）that the CDOCKER interaction energy of LF and TRPC1 was―86.03 kcal/mol.LF formed hydrogen bond interaction with LYS 248,LYS 301,GLN 517,GLN 514,LYS 648,LYS 297.These hydrogen bonding interactions promoted the stable binding of small molecules to the protein active site.The binding constant Kd of LF to TRPC1 protein was 41.23±12.45μM,indicating that LF was directly bound to TRPC1.The results of q PCR and Western blot suggested that LF could reduce the Trpc1 m RNA and protein levels by TGF-β₁-stimulated and inhibit the Ca²⁺extracellular influx by TGF-β₁-stimulated liver fibrosis injury in LX-2 cells.（3）LF can improve the CCl₄-induced liver fibrosis in miceCompared to the CCl₄ group,the ALT activity in the serum of the CCl₄+H-LF group decreased by 81%,the AST activity decreased by 66%,the Alb activity increased by 81%,in liver tissue the activity of SOD increased by 68%,the concentration of GSH-Px increased by 34%,the concentration of GSH-Px decreased by 59%,and the content of Hyp decreased by 30%,suggesting that high-dose LF group can improve CCl₄-induced liver function and oxidative damage in mice.H&E staining showed that hepatocyte cords were neatly arranged,fibrous tissue proliferation and inflammatory cell infiltration decreased significantly in CCl₄+H-LF group,and Sirius Red staining and Masson staining showed that CCl₄+H-LF group could improve liver fibrosis and bridging fibrosis.Immunohistochemical staining showed that compared to the CCl₄ group,the expression consistency ofα-SMA and Col1a1protein in the central vein area,hepatic sinusoid space and portal area significantly decreased in the CCl₄+H-LF group.Western blot results showed that the expression ofα-SMA and Col1a1 protein also decreased by 45%and 53%,respectively.The immunofluorescence results showed that compared to TGF-β₁ group,the positive area ofα-SMA and Col1a1 expression in TGF-β₁+LF group decreased by 97%and 1.1-fold,respectively.Western blot results also confirmed that the protein expression ofα-SMA and Col1a1 in TGF-β₁+LF group decreased by 1.1-fold and 75%,respectively.The above results suggested that LF could significantly inhibit HSCs activation and ECM deposition in vivo and in vitro.In addition,LF could significantly inhibit the expression of TβRⅠ,TβRⅡ,Smad4,p-Smad2/3 protein up-regulated in TGF-β₁-stimulated LX-2 cells.5 Conclusion（1）TRPC1 was a potential new drug target for intervention of liver fibrosis.TRPC1 was highly expressed in both human and mouse liver fibrosis,and mainly expressed in activated HSCs;after Trpc1 gene knockout in vivo and Trpc1 gene silencing in vitro,they could inhibit TGF-β/Smad signal pathway,down-regulate the expression of Smad2/3phosphorylated protein,reduce the expression ofα-SMA and Col1a1 protein,and then slow down the occurrence and development of liver fibrosis;Trpc1 gene silencing could inhibit TGF-β₁ activating SOCE in HSCs.（2）LF,the active component of Erzhi pill,had potential anti-fibrotic activity.LF had a strong affinity with TRPC1,inhibited the expression and function of TRPC1 in TGF-β₁-stimulated HSCs,inhibited the activation of TGF-β/Smad pathway,alleviated CCl₄-induced liver fibrosis in mice.