Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.

The mechanism of Yinchenhao decoction in treating obstructivejaundice- induced liver injury based on Nrf2 signaling pathway

BACKGROUND Obstructive jaundice(OJ)is caused by bile excretion disorder after partial or complete bile duct obstruction.It may cause liver injury through various mechanisms.Traditional Chinese medicine(TCM)has a lot of advantages in treating OJ.The recovery of liver function can be accelerated by combining Chinese medicine treatment with existing clinical practice.Yinchenhao decoction(YCHD),a TCM formula,has been used to treat jaundice.Although much progress has been made in recent years in understanding the mechanism of YCHD in treating OJ-induced liver injury,it is still not clear.AIM To investigate chemical components of YCHD that are effective in the treatment of OJ and predict the mechanism of YCHD.METHODS The active components and putative targets of YCHD were predicted using a network pharmacology approach.Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes path enrichment analysis were carried out by cluster profile.We predicted the biological processes,possible targets,and associated signaling pathways that YCHD may involve in the treatment of OJ.Thirty male Sprague–Dawley rats were randomly divided into three groups,each consisting of 10 rats:the sham group(Group S),the OJ model group(Group M),and the YCHDtreated group(Group Y).The sham group only received laparotomy.The OJ model was established by ligating the common bile duct twice in Groups M and Y.For 1 wk,rats in Group Y were given a gavage of YCHD(3.6 mL/kg)twice daily,whereas rats in Groups S and M were given the same amount of physiological saline after intragastric administration daily.After 7 d,all rats were killed,and the liver and blood samples were collected for histopathological and biochemical examinations.Total bilirubin(TBIL),direct bilirubin(DBIL),alanine aminotransferase(ALT),and aspartate transaminase(AST)levels in the blood samples were detected.The gene expression levels of inducible nitric oxide synthase(iNOS)and endothelial nitric oxide synthase(eNOS),and the nucleus positive rate of NF-E2 related factor 2(Nrf2)protein were measured.Western blot analyses were used to detect the protein and gene expression levels of Nrf2,Kelchlike ECH-associated protein 1,NAD(P)H quinone dehydrogenase 1(NQO1),and glutathione-Stransferase(GST)in the liver tissues.One-way analysis of variance was used to evaluate the statistical differences using the statistical package for the social sciences 23.0 software.Intergroup comparisons were followed by the least significant difference test and Dunnett’s test.RESULTS The effects of YCHD on OJ involve biological processes such as DNA transcription factor binding,RNA polymerase II specific regulation,DNA binding transcriptional activator activity,and nuclear receptor activity.The protective effects of YCHD against OJ were closely related to 20 pathways,including the hepatitis-B,the mitogen-activated protein kinase,the phosphatidylinositol 3-kinase/protein kinase B,and tumor necrosis factor signaling pathways.YCHD alleviated the swelling and necrosis of hepatocytes.Following YCHD treatment,the serum levels of TBIL(176.39±17.03μmol/L vs 132.23±13.88μmol/L,P<0.01),DBIL(141.41±14.66μmol/L vs 106.43±10.88μmol/L,P<0.01),ALT(332.07±34.34 U/L vs 269.97±24.78 U/L,P<0.05),and AST(411.44±47.64 U/L vs 305.47±29.36 U/L,P<0.01)decreased.YCHD promoted the translocation of Nrf2 into the nucleus(12.78±0.99%vs 60.77±1.90%,P<0.001).After YCHD treatment,we found a decrease in iNOS(0.30±0.02 vs 0.20±0.02,P<0.001)and an increase in eNOS(0.18±0.02 vs 0.32±0.02,P<0.001).Meanwhile,in OJ rats,YCHD increased the expressions of Nrf2(0.57±0.03 vs 1.18±0.10,P<0.001),NQO1(0.13±0.09 vs 1.19±0.07,P<0.001),and GST(0.12±0.02 vs 0.50±0.05,P<0.001),implying that the potential mechanism of YCHD against OJ-induced liver injury was the upregulation of the Nrf2 signaling pathway.CONCLUSION OJ-induced liver injury is associated with the Nrf2 signaling pathway.YCHD can reduce liver injury and oxidative damage by upregulating the Nrf2 pathway.

Effects of Yinchenhao Decoction on Self-regulation of Renin-angiotensin System by Targeting Angiotensin Converting Enzyme 2 in Bile Duct-ligated Rat Liver

Summary： In order to investigate whether Yinchenhao decoction （YCHD） attenuates hepatic fibro- genesis in the bile duct ligation （BDL） model via recovering and restoring the self-regulation and bal- ance of the renin-angiotensin system （RAS）, 33 specific-pathogen-free （SPF） male Sprague-Dawley rats with common BDL and scission were randomly divided into five groups as follows： G1, the sham group （n=4）; G2, BDL 7-day group （n=5）; G3, BDL＋YCHD 430 mg/mL （n=8）; G4, BDL＋losartan 0.65 mg/mL （ARB group, n=8）; G5, model group （BDL without any treatment, n=8）. YCHD and losartan （10 mL.kgl.day-1） were given by gastric gavage for 16 days following BDL in G3 and G4 groups, respec- tively. The effect of YCHD on liver fibrosis and the detailed molecular mechanisms were assessed by liver function including total bilirubin （TBIL）, direct bilirubin （DBIL）, indirect bilirubin （IDBIL）, alanine aminotransferase （ALT）, and aspartate aminotransferase （AST）. Histological changes were ob-. served by transmission electron microscopy （TEM） and Masson trichrome staining. Western blotting was used to detect the protein expression level of the renin-angiotensin system （RAS） components in- cluding angiotensin converting enzyme （ACE）, angiotensin II type 1 receptor （AT1R）, ACE2, angio- tensin II （Ang II） as well as transforming growth factor 131 （TGF131）. The experimental data were ana- lyzed by principle component analytical method of pattern recognition. The results showed that bio- chemically, serum TBIL, DBIL, IDBIL, ALT and AST levels were markedly increased following BDL as compared with the sham group （P〈0.05）. Serum TBIL, IDBIL and DBIL levels in G3 group were dramatically decreased as compared with G5 and G4 groups （P〈0.05）. Serum AST level in G3 was sig- nificantly lowered than in G5 group （P〈0.05）, but there was no significant difference in ALT among G3, G4 and G5 groups （P〉0.05）. Histologically, livers in G3 group showed less hepatocytes necrosis, less bile duct hyperplasia and less collagen formation than in G4 and G5 groups. The protein expression lev- els of ACE2, ACE, Ang II, AT1R and TGF131 in G2, G3 and G4 groups were significantly higher than in sham group （P〈0.05）, and lower than in G5 group （P〈0.05）. However, the differences among G2, G3 and G4 groups were not significant （P〉0.05）. ACE2 protein expression in G3 group was significantly higher than in G2 group （P〈0.05） and there was no significant difference in comparison with G4 group （P〉0.05）. Moreover, the protein expression of TGF131 in G3 group was significantly lower than in G5 and G4 groups （P〈0.05）. Our findings suggest that the antifibrotic effects of YCHD may be associated with the decreased classical RAS pathway components and TGFI31 downexpression so as to recover and rebuild self-regulation of the RAS by elevating the protein expression of ACE2.

Mechanism of Yinchenhao decoction in the treatment of jaundice based on network pharmacology

Objective:This study aimed to examine the mechanism of classic ancient prescription of Chinese medicine Yinchenhao decoction in treating jaundice based on network pharmacology.Method:An oral bioavailability of≥30%,a drug likeness of≥0.18,and literature studies were used to screen for Yinchen(Artemisiae scopariae herba),Zhizi(Gardeniae fructus),Dahuang(Rhei radix et rhizome)in the Chinese Medicine System Pharmacology Database and Analysis Platform.The active ingredient was introduced into the PubChem database to collect drug component targets and import into the Uniprot database for gene standardization.The target gene of Yinchen(Artemisiae scopariae herba)was screened via Human Gene Database(GeneCards).Then,use the Cytoscape 3.7.2 software was used for network visualization analysis,and the R3.6.1 software was used for gene ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses.Results:We collected a total of 47 active constituents of classic ancient prescription of Chinese medicine Yinchenhao decoction,of which 17 were related to jaundice;189,9 targets of jaundice were screened,of which 41 were interdigitated with the targets of classic ancient prescription of Chinese medicine Yinchenhao decoction.Gene ontology functional enrichment analysis revealed 111 biological processes,14 cellular components,and 28 molecular functions,and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed 34 Kyoto Encyclopedia of Genes and Genomes pathways including hepatocellular carcinoma,PI3K-Akt signaling pathway,HIF-1 signaling pathway,prolactin signaling pathway,and non-alcoholic fatty liver disease.Conclusion:Based on the network pharmacology,the analysis of jaundice and classic ancient prescription of Chinese medicine Yinchenhao decoction provides a novel idea and direction for the study of classic ancient prescription of Chinese medicine Yinchenhao decoction in the treatment of jaundice.

Immunomodulation and liver protection of Yinchenhao decoction against concanavalin A-induced chronic liver injury in mice

OBJECTIVE：This study investigated the immunoregulatory and protective roles of Yinchenhao decoction,a compound of Chinese herbal medicine,in a mouse model of concanavalin A（Con A）-induced chronic liver injury.METHODS：Female Bal B/c mice were randomly divided into 4 groups：normal control,Con A model,Con A model treated with Yinchenhao decoction（400 mg/kg,orally）,and Con A model treated with dexamethasone（0.5 mg/kg,orally）.All treatments were given once a day for 28 d.Except of the normal control,mice received tail vein injection of Con A（10 mg/kg）on days 7,14,21,and 28,at 1 h after treatment with Yinchenhao decoction or dexamethasone or saline to induce chronic liver injury.RESULTS：Repeated Con A injection induced chronic liver injury,which was evidenced by infl ammatory cell infi ltration and necrosis,increased serum alanine aminotranferease activities,decreased albumin levels,and an imbalanced expression of immunoregulatory genes in the liver tissues including signifi cantly enhanced interferon-γ,interleukin-4,monocyte chemotactic protein-1,and cluster of differentiation 163 m RNA levels,and reduced tumor necrosis factor-αand interleukin-6 m RNA levels.Treatment with Yinchenhao decoction signifi cantly reversed the Con A-induced changes in immunoregulatory gene expression in the liver tissues,reduced serum alanine aminotranferease activity,enhanced serum albumin level,and attenuated the extent of liver infl ammation and necrosis.Furthermore,Yinchenhao decoction did not result in hepatocyte degeneration and spleen weight loss that were observed in mice received long-term treatment with dexamethasone.CONCLUSION：Yinchenhao decoction treatment protected liver against the Con A-induced chronic liver damage and improved liver function,which were associated with the modulation of gene expression related to immune/infl ammatory response.

Comparative Study on the Protective Effects of Yinchenhao Decoction(茵陈蒿汤) against Liver Injury Induced byα-Naphthylisothiocyanate and Carbon Tetrachloride

Objective:To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine.Methods:The liver injury in the model rat was induced byα-naphthylisothiocyanate(ANIT) and carbon tetrachloride(CCl_4) respectively,and the effects of Yinchenhao Decoction(茵陈蒿汤,YCHD),a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic,on the two liver injury models were evaluate...

Changes of Pharmacokinetics of 6,7-Dimethoxycoumarin in A Rat Model of Alpha-Naphthylisothiocyanate-Induced Experimental Hepatic Injury after Yinchenhao Decoction(茵陈蒿汤) Treatment

Objective： To study the changes of pharmacokinetics of 6,7-dimethoxycoumarin in a rat model of alpha-naphthyiisothiocyanate （ANIT）-induced experimental hepatic injury after oral administration of Yinchenhao Decoction （茵陈蒿汤, YCHD） using an ultra pressure liquid chromatography （UPLC） method. Methods： Rats were divided into a normal group and a model group, after modeled by 4% ANIT （75 mg/kg） for 48 h, they were orally administrated with YCHD extract at the dose of 0.324 g/kg, and then blood was collected from their orbital sinus after different intervals. Changes in liver function were monitored by the levels of liver enzymes [alanine aminotransferase （ALl＇）, aspartate aminotransferase （AST）] and bilirubins [total bilirubin （TBIL）, direct bilirubin （DBIL）], the concentration of 6,7-dimethoxycoumarin in plasma were measured by UPLC, and the pharmaceutical parameters were calculated with DAS2.1.1 software. Results： The concentration-time curve of both normal and modeled rats after oral administration of YCHD was obtained. Their time to maximum plasma concentration （tmax were both 0.25 h, the maximum concentration （Cmax） were 4.533 iμ g/mL and 6.885 μg/mL, and their area under concentration-time curve （AUC）o→24h were 16.272 and 32.981, respectively. There was a 51.88% and 100.46% increase in Cmax and AUCo-t （P〈0.05）, but there showed a 45.52% and 92.93% reduction in clearance of drug and volum of distribution （P〈0.05）, respectively. Conclusions： Hepatic injury could significantly influence the pharmacokinetics of 6,7-dimethoxycoumarin after oral administration of YCHD, the absorption and distribution process was accelerated in liver injured rats, but the metabolism and elimination process was slowed. And this may lead to a significant accumulation of 6,7-dimethoxycoumarin in the body.

Effects of Yinchenhao Decoction(茵陈蒿汤) for Non-alcoholic Steatohepatitis in Rats and Study of the Mechanism

Objective:To observe the effects of Yinchenhao Decoction(茵陈蒿汤) for non-alcoholic steatohepatitis(NASH) in rats and study the mechanism.Methods:Total 18 male SD rats were randomly divided into a normal control group,a model group and a treatment group,6 rats in each group.Rats in the model and treatment groups were fed with high-fat forage for 10 weeks to prepare the NASH model,and the rats in the treatment group were administrated with Yinchenhao Decoction from the 6th week for 5 weeks.All rats were sacrificed at the end of the10th week and the samples were collected.Serum alanine aminotransferase(ALT) activity,tumor necrosis factor-α(TNF-α) level,and hepatic triglyceride(TG) and free fatty acid(FFA) contents were determined.Hepatic pathological changes were detected by HE staining.Results:Serum ALT activity,TNF-α level,hepatic TG and FFA contents,and the fatty deposition in hepatocytes were significantly reduced in the rats of the treatment group.Conclusion:Yinchenhao Decoction has good therapeutic effects for NASH,protecting the liver function and reducing the fatty deposition in liver,which are possibly related with reduction of FFA content and inhibition of TNF-α expression.

Rational Daily Administration Times of Yinchenhao Decoctionin Rats with Jaundice Based on PD/PK

Objective To study the rational daily administration times of Yinchenhao Decoction(YCHD)when it was used to treat experimental jaundice in rats based on pharmacodynamics/pharmacokinetics model.Methods Rats were modeled by 4%1-naphthylisothiocyanate(75 mg/kg)for 48 h,then YCHD was drenched with doses of 0.324 g/kg (extract,calculated with the clinical dosage)once,0.162 g/kg twice,and 0.108 g/kg thrice a day,respectively.The total bile and the flow rate of bile were observed after the first administration;Blood samples collected from the orbital sinus at different intervals were used to investigate the levels of liver enzymes(ALT and AST)and bilirubins (TBIL and DBIL),and determine the concentration of 6,7-dimethoxycoumarin(DME)in the plasma using UPLC at the same time,then we obtained the time-effect and time-dose curves.The rational daily administration times of YCHD when treating experimental jaundice were determined based on the comprehensive analysis of time-effect and time-concentration relationships.Results Within 10 h the total bile of rats which were administered once daily(G1) was 1.65 and 1.33 times higher than that of twice and thrice(G2 and G3)a day,respectively,and the four biochemical indexes(TBIL,ALT,DBIL,and AST)of G1 decreased faster than those of G2 and G3(P<0.05).On the other hand, the blood drug level of DME when administrated once daily could maintain at a higher level for a longer time,and its Cmax and AUC0→t were higher than those of G2 and G3,which might be the main reason why its effect was the most significant.Conclusion It is more appropriate to administrate once daily when YCHD is used to treat jaundice.

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The International Society for Ethnopharmacology is a collaborative, interdisciplinary group of scientists–anthropologists, pharmacists, pharmacologists, ethnobotanists, phytochemists, and others–all fascinated by the study of the local and global use of traditional medicines. Traditional medicinal knowledge has been transferred over