Pharmacodynamic Evaluation And Preliminary Mechanism Of Yinhuang Granule In Improving Pulmonary Fibrosis And Liver Fibrosis In Mice

Background:Fibrosis is a pathological process during which tissue and organs repair themselves after injury,resulting in the abnormal increase and excessive deposition of extracellular matrix（ECM）.It can occur in a variety of organs,and the continuous progress will lead to the destruction of organ structure and functional failure with the serious will cause harm to human health and life.Pulmonary fibrosis and hepatic fibrosis are two common diseases of organic fibrosis.Yinhuang granule（YHG）,composed of Scutellariae Radix and Lonicerae Japonicae Flos,has the effect of clearing away heat and detoxification.YHG is often used to treat sore throat,dry pharynx,acute and chronic pharyngitis and upper respiratory tract infection in clinic.There is still no report about the amelioration provided by YHG on organic fibrosis.Objective:This study aims to explore the alleviation of YHG on pulmonary fibrosis and liver fibrosis,and further to explore the engaged mechanism through network pharmacology analysis combined with experimental verification.Our experiments will provide experimental basis for the application of YHG in the prevention and treatment of pulmonary fibrosis or liver fibrosis,and also provide new ideas for the clinical treatment and drug development for pulmonary fibrosis or liver fibrosis.Methods:1.Network pharmacology analysis:The effective compounds and their related targets of Scutellariae Radix and Lonicerae Japonicae Flos were screened out by traditional Chinese medicine systems pharmacology database and analysis platform（TCMSP）database.Gene symbol corresponding to the effective target was screened out by Uni Prot protein database.Data were then imported into the software cyberscape 3.7.2for visualization,and the effective compound of YHG-target information map was obtained.In GENECARD database,the disease-related targets were searched by using the keywords:pulmonary fibrosis or liver fibrosis.Wayne map was made after combined the disease targets and drug targets to obtain these co-owned targets.The interaction path map was obtained by substituting those above targets into string database.The molecular function（MF）,biological process（BP）,cell component（CC）and KEGG pathway were obtained by META scape analysis of GO gene enrichment and KEGG pathway,and thus the potential targets involved in the YHG-provided amelioration on pulmonary fibrosis or liver fibrosis were obtained.2.YHG alleviated pulmonary fibrosis in vivo:Bleomycin（BLM）（Intraperitoneal injection,i.p.）was used to induce pulmonary fibrosis in mice,and different doses of YHG（400,800mg/kg）were orally given to mice.The lung function of mice was detected by lung function ventilator.The lung injury was observed by hematoxylin eosin staining（H&E）.Sirius red and Masson’s trichrome staining were used to observe the collagen deposition in lung tissues.The levels of collagen I,collagenⅢand hydroxyproline（HYP）were detected by using enzyme linked immunoassay（ELISA）.The protein concentration in bronchoalveolar lavagefluid（BALF）was measured by using BCA kits.Myeloperoxidase（MPO）activity was detected by using kits.Real-time PCR（RT-PCR）was used to detect the m RNA expression ofα-smooth muscle actin（α-SMA）and transforming growth factorβ（TGF-β）.3.YHG alleviated liver fibrosis in vivo:Bile duct ligation（BDL）,carbon tetrachloride（CCl₄）（i.p.,2 m L/kg）and methionine/choline deficiency diet（MCD）were used to induce liver fibrosis in mice,and different doses of YHG（400,800mg/kg）were orally given to mice.Serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）activities were detected.Hepatic HYP content was detected by using kits.Liver injury was evaluated by H&E staining.Hepatic collagen deposition was observed by using Sirius red and Masson staining assay.RT-PCR was used to detect hepatic m RNA expression of molecules related with fibrosis such as COL1A1,COL3A1,α-SMA,FN1and TGF-β,molecules related with the antioxidant responses such as NAD（P）H quinone oxidoreductase 1（NQO1）,heme oxygenase 1（HO-1）,glutamic acid cysteine ligase catalytic subunit（GCLC）,gluamate cystein ligase modifier subunit（GCLM）and superoxide dismutase（SOD）,and molecules related with inflammation including interleukin-1 beta（IL-1β）,cyclooxygenase-2（COX-2）,tumor necrosis factor alpha（TNF-α）and inducible nitric oxide synthase（i NOS）.Serum contents of hyaluronic acid（HA）and collagen IV（COLIV）were detected by using ELISA kits.Hepatic MPO and SOD activity,and liver contents of reactive oxygen species（ROS）,protein carbonylation（PC）,glutathione（GSH）,malondialdehyde（MDA）and adenosine triphosphate（ATP）were detected by using kits.The nuclear accumulation of Nrf2 and the expression of its down-stream molecules including GCLC,GCLM and NQO1 were detected by using Western-blot assay.Result:1.A total of 23 compounds were screened out from Lonicerae Japonicae Flos,among which 17 compounds with target points,and total 400 target information was found.A total of 36 compounds were screened out from Scutellariae Radix,among which 32compounds with target points,and total 465 target information was found.There are1924 important targets for pulmonary fibrosis,among which total 123 co-owned targets for both pulmonary fibrosis and YHG.There are 1597 targets for liver fibrosis,among which total 125 co-owned targets for both liver fibrosis and YHG.The results of enrichment analysis showed that YHG can improve pulmonary fibrosis or liver fibrosis,which may be related to inhibit inflammation,alleviate oxidative stress injury and reduce ECM deposition.2.YHG rescued the decrease of lung function,decreased the elevated serum contents of COL1A1,COL3A1 and HYP,decreased the enhanced protein concentration of BALF,decreased the increased MPO activity,reduced the elevated m RNA expression of fibrosis related molecules and decreased the deposition of collagens in lung tissues from mice treated with BLM.3.YHG attenuated liver injury induced by BDL,CCl₄ and MCD in mice.YHG reduced the increased hepatic expression of HYP and fibrosis related molecules,reduced the enhanced hepatic deposition of collagens and reduced the activation of hepatic stellate cell（HSC）.YHG alleviated liver oxidative stress injury through inducing Nrf2activation and increasing the expression of its downstream antioxidant molecules in mice treated with CCl₄ or MCD.YHG also attenuated liver inflammatory injury via reducing the enhanced expression of pro-inflammatory cytokines in mice treated with MCD.Conclusions:YHG can alleviate pulmonary fibrosis or liver fibrosis in vivo.The results from network pharmacology and the following experiments implied that YHG attenuated pulmonary fibrosis or liver fibrosis through inhibiting inflammation and oxidative stress injury,and reducing the deposition of ECMs.