Research On The Effect Of Dahuangzhechong Pills And The Effective Substance Monomer Kaempferol In Preventing Hepatic Fibrosis And The Mechanism Related To ASIC1a/VEGF

Hepatic fibrosis（HF）is a dynamic process of persistent wound healing,and its pathogenesis is due to liver injury caused by a variety of factors.It is the basic stage of various liver diseases,including cirrhosis and liver cancer,and is a potential threat to human health and a huge disease burden worldwide,including in China.Hepatic stellate cells（HSC）are non-parenchymal cells that exist in the liver.The overproduction of extracellular matrix（ECM）is an important feature of HSC activation,and the accumulation of ECM compresses blood vessels,causing local hypoxia and creating a prerequisite for the release of pro-fibrotic factors such as vascular endothelial growth factor（VEGF）.This creates a prerequisite for the release of VEGF and other pro-fibrotic factors.It is well documented that HSC enhances the interaction with surrounding cells through the secretion of VEGF,remodeling the liver and exacerbating the substantial liver damage during the HF process.Acid-sensing ion channels（ASICs）are non-selective cation channels that are activated by extracellular acidification,i.e.,increased H+.Acid-sensing ion channel1a（ASIC1a）is one of the most studied isoforms in the ASICs family.When extracellular p H decreases,the channel activates and mediates Ca²⁺ inward flow,participating in a variety of physiological and pathological processes in the body.Previous studies by our group have shown that early liver fibrosis is accompanied by local hypoxia,and local tissue acidification caused by hypoxia enhances the ability of ASIC1 a to mediate Ca²⁺ inward flow,causing intracellular Ca²⁺ overload and internal homeostatic imbalance,triggering endoplasmic reticulum stress（ERS）to play an important role in liver diseases such as liver failure and liver cancer.The ERS plays an important role in liver failure,liver cancer and other liver diseases.Our group further found that ERS can promote the activation of HSC to accelerate the development of HF,and this process is regulated by ASIC1 a.Zhang Zhongjing,a Han Dynasty medical scientist,elaborated the whole formula of Dahuangzhechong pills（DHZCP）in his book "Jin Kui Yao Lve".The whole formula includes a variety of active pharmaceutical ingredients,which have been proven by modern medicine to have clear therapeutic effects in the treatment of HF.However,the complex composition of the compound formula makes it difficult to explain the effects of different flavor combinations among the whole formula by traditional pharmacological methods.In combination with previous studies,we obtained Kaempferol,one of its active substance monomers,which belongs to flavonoid alcohol structure and has been shown to have anti-tumor,anti-inflammatory and anti-lipid metabolism disorder effects.However,the effects on HSC activation and VEGF release and the underlying molecular mechanisms have not been clearly elucidated.To this end,this project clarifies the therapeutic effect of compound DHZCP on the overall rat HF model from in vivo experiments,and initially investigates the regulatory effect of DHZCP on ASIC1a/VEGF pathway.And through in vitro experiments,rat HSC-T6 cells were used to further investigate the anti-HF effect and inhibition of ASIC1a/ERS/VEGF pathway by its active ingredient Kaempferol,which can provide new ideas for the study of HF pathogenesis as well as new drug development.The main study design is as follows:1.The effect of DHZCP on HF in vivoA rat HF model was established by intraperitoneal injection of CCl4 mixed with olive oil solution.And the rats were randomly divided into control group,model group,DHZCP treatment group（low,medium and high dose）and positive control drug colchicine group.The degree of liver injury in each group was evaluated by HE staining,serological indexes,and Masson staining to observe collagen deposition and fibrous stripe production in each group.Immunohistochemistry,q RT-PCR and Western Blot were used to detect the m RNA and protein levels of ASIC1 a,α-SMA,Collagen-I,Ca MMKβ and VEGF in the liver tissues of each group.The results showed that DHZCP could effectively alleviate HF,and the administration of each dose group reversed the elevated m RNA and protein levels of ASIC1 a,α-SMA,Collagen-I,Ca MMKβ and VEGF in liver tissues of the model group.It is suggested that DHZCP can alleviate HF,and the mechanism may be related to ASIC1a/VEGF.2.Screening of active monomer Kaempferol and its effect on HSC-T6 activation and VEGF release in vitroThe 89 active ingredients in DHZCP obtained in the previous stage were combined with docking scores and the effects of each representative ingredient on α-SMA and VEGF proteins were detected by q RT-PCR,Western Blot method to obtain the preliminary screening of monomeric Kaempferol.subsequently,we used CCK-8 method to detect the toxic safety range of Kaempferol.Time gradients（6h,12 h,24h,48h）and concentration gradients（5μM,10μM,20μM）were set to detect the expression levels of α-SMA,Collagen-I and VEGF in HSC-T6 cells by q RT-PCR and Western Blot assay.The effect of Kaempferol on VEGF expression was detected by immunofluorescence assay.The results showed that Kaempferol was non-toxic to cells in the range of 20 μM and inhibited HSC-T6 activation and VEGF release in a time-and dose-dependent manner.3.Effect of Kaempferol-targeted binding to ASIC1 a on promoting ASIC1 a protein degradationMolecular docking,CETSA assay was used to detect the interaction between Kaempferol and ASIC1 a protein.The results showed that Kaempferol could form hydrogen bond binding to ASIC1 a protein structure and improved its thermal stability,and there was a certain binding effect.q RT-PCR,Western Blot,and immunofluorescence were used to detect the effect of Kaempferol on ASIC1 a protein.The results showed that the ASIC1 a protein level was decreased by Kaempferol treatment and the m RNA level was basically unchanged,indicating that the decrease of ASIC1 a protein level by Kaempferol may involve the degradation pathway.The specific degradation pathway was further determined by Western Blot method using the tool drug cycloheximide（CHX）as well as inhibitors of each degradation pathway.The results suggest that the promotion of ASIC1 a protein degradation by Kaempferol may be related to the ubiquitination pathway.4.Effect of Kaempferol on intracellular Ca²⁺ concentration and ERS levelHSC-T6 cells were pretreated by Kaempferol and the changes of intracellular transient Ca²⁺ concentration under p H 6.0 stimulation were detected by laser confocal microscopy.Western Blot was also used to detect the effect of Kaempferol on calcium-regulated kinase,which indirectly reflects the change of intracellular Ca²⁺ level.In addition.The intracellular Ca²⁺ aggregation was detected using endoplasmic reticulum and mitochondrial probes,and the changes of intracellular ERS in HSC-T6 cells after Kaempferol treatment were further investigated by q RT-PCR,Western Blot.The results showed that after Kaempferol treatment the intracellular Ca²⁺ in HSC-T6 cells inward flow was inhibited and the expression of p-e IF2α,ATF4 and VEGF proteins in ERS was reduced.5.Effect of Kaempferol on ASIC1 a,HSC-T6 activation,p-e IF2α,ATF4 and VEGF protein expression in ERS after inhibition of ASIC1 a activity or overexpression of ASIC1aThe effect of Kaempferol on the expression of ASIC1 a,α-SMA,Collagen-I,and p-e IF2α,ATF4,and VEGF proteins in ERS was detected by Western Blot after inhibition of ASIC1 a activity using the ASIC1a-specific inhibitor Pc Tx-1.In addition,overexpression of ASIC1 a was performed using overexpression plasmids,q RT-PCR,and Western Blot to detect the above proteins.The results showed that Kaempferol further reduced ASIC1 a protein levels on top of ASIC1 a inhibition by Pc Tx-1;after ASIC1a overexpression,the inhibitory effect of Kaempferol was diminished.It is suggested that the action of Kaempferol may be achieved by inhibiting ASIC1 a.6.Enhancement of ERS by pretreatment with toxic carotene（TG）and observation of the effect of Kaempferol on ERS-related protein and HSC-T6 cell activation and VEGF protein expressionTG pretreated HSC-T6 cells were agitated with ERS for 1h.Kaempferol was co-incubated with PDGF-stimulated HSC-T6 cells for 24 h,and the expression of ERS-related proteins p-e IF2α,ATF4 and α-SMA,Collagen-I and VEGF were detected.The results showed that the inhibitory effect of Kaempferol on α-SMA,Collagen-I and VEGF expression was diminished after ERS agitation.The above results suggest that Kaempferol may inhibit HSC-T6 cell activation and VEGF release through the ERS pathway.