Effects of Yigan Capsule on the expression of HMGB1, RAGE and NFκB protein in rats with drug-induced liver injury

TANG Ya, LI Jun, QI Yazhi, CAO Rui, ZHAI Yan-ling, HAN Yu-sheng?, XU Qiang?

1. Heilongjiang University of Tradit Chin Med, Harbin 150040, China

2. Heilongjiang University of Chinese Medicine Jiamusi College, Jiamusi 154007,China

Keywords:

ABSTRACT Objective: To study the effect of Yigan capsule on the expression of high mobility group protein B1 (HMGB1), nuclear factor-B (NF-κB)and receptor for advanced glycation end products ( RAGE )in anti-tuberculosis drug-induced liver injury (ATB-DILI), and to explore its protective effect and mechanism on ATB-DILI, so as to provide experimental basis for the clinical application of Yigan capsule.Methods: Twenty-four rats were divided into two groups.Except for the blank group (n = 6), the other 18 rats were given isoniazid (INH)+ rifampicin(RFP)(50 mg/kg.d)for 4 weeks.Then 18 rats were randomly divided into three groups (model group, low dose group of Yigan capsule and high dose group of Yigan capsule)according to 6 rats in each group.The blank group and the model group were given 0.9 % sodium chloride solution by intragastric administration.The low dose group of Yigan capsule was 0.468 g / kg,and the high dose group of Yigan capsule was 1.872 g / kg [1].After 4 weeks, the pathological changes of liver were observed by HE staining.The contents of ALT, AST, ALP, γ-GT and TBIL were detected.The expression of HMGB1, NF-κBp65 and RAGE protein was detected by IHC.The expression levels of HMGB1, NF-κBp65, RAGE, TNF-α and IL-1β were detected by WB.Result: HE staining showed that the structure of the liver in the model group was disordered, the liver cells showed swelling and fusion, the number of inflammatory cells increased and accompanied by punctate necrosis, while the above pathological changes in each treatment group of Yigan capsule were significantly improved.The contents of ALT,AST, ALP, γ-GT and TBIL in the model group were higher than those in the blank group(P＜ 0.05).The contents of ALT, AST, ALP, γ-GT and TBIL in each treatment group were significantly lower than those in the model group (P＜0.05).Compared with the blank group,the expression levels of TNF-α and IL-1β in the model group were increased (P＜0.05), and the expression levels of HMGB1, NF-κBp65 and RAGE were increased (P＜0.05).Compared with the model group, the expression levels of TNF-α and IL-1β in each treatment group of Yigan capsule decreased (P＜0.05), and the expression of HMGB1, NF-κBp65 and RAGE decreased (P＜0.05).Conclusion: Yigan capsule may inhibit the secretion of inflammatory factors through HMGB1/RAGE/NF-κBp65 signaling pathway, thus protecting ATB-DILI.

1.Introduction

As the second largest single source of infection in the world,tuberculosis is second only to coronavirus disease 2019 (COVID-19)in terms of mortality[2].Anti-tuberculosis drug-induced liver injury (ATB-DILI)is a high incidence of tuberculosis treatment adverse reactions[3].Studies have shown that high mobility group protein B1(HMGB1)is a key protein that mediates the pathogenesis of chronic and acute liver diseases, including APAP-induced drug-induced liver injury (DILI), non-alcoholic fatty liver disease(NAFLD), HBV-induced liver disease, liver fibrosis, liver cancer and acute liver failure[4],HMGB1 and receptor for advanced glycation end products RAGE)are widely expressed in liver tissue [5].When liver cells are damaged, a large amount of released HMGB1 will induce the secretion of pro-inflammatory cytokines and the aggregation of immune cells, so that the inflammatory response continues to occur, which further aggravates liver injury[4].

At present, western medicine has no effective means for the prevention and treatment of ATB-DILI, Tradit Chin Med has the characteristics of multi-component, multi-target and few side effects,and has unique advantages in clinical prevention and treatment of ATB-DILI.Yigan capsule is an empirical prescription for the treatment of liver injury, which has a significant clinical effect on the prevention and treatment of liver injury, previous experimental studies have shown that it has anti-inflammatory, inhibition of HMGB1 protein expression and anti-lipid peroxidation effects[7,6].In this study, isoniazid (INH)and rifampicin (RFP)were used to replicate the rat model of ATB-DILI, to study whether Yigan Capsule has a protective effect on ATB-DILI through HMGB1/RAGE/NFκB signaling pathway, and to provide experimental basis for clinical application.

2.Materials and methods

2.1 Experimental animal

SPF grade, SD male mice, body weight (160～180) g, Liaoning Changsheng Biotechnology Co., Ltd, License No.: SCXK(Liao)2020-0001.The experiment began after one week of feeding.

2.2 Medicine and reagent

Guangdong Huanan Pharmaceutical Isoniazid Tablets (H44020699),Rifampicin Capsules (H44020771); Kushen,Danshen,Wuweizi according to the ratio of 1:3:3, the preparation of Yigan Capsule(medicinal materials purchased from Beijing Tong ren tang Pharmacy Harbin Branch);ALT (Batch No.:C009-2-1),AST(Batch No.:C010-2-1),ALP (Batch No.: A059-2-2), γ-GT(Batch No.:C017-2-1), TBIL (Batch No.:C019-1-1)detection kit(Nanjing Jiancheng Biotechnology Co.,Ltd.);Rabbit anti-TNFα(bs-10802R),rabbit anti-IL-1 (bs-0812R), rabbit anti-RAGE (bs-0177R), rabbit anti-Actin (bs-0061R)(Beijing Boaosen);Rabbit anti-NF-κBp65 (bs-0664R)(Boster);rabbit anti-HMGB1 (P09429)(Jiangsu pro-family biology);rabbit two-step kit (PV-6001)(Beijing Zhongshan Jinqiao);Anti-rabbit IgG-HRP (bs-0295G-HRP),DAB chromogenic agent, pre-stained broad-spectrum protein Marker(Beijing Boaosen);BCA kit, electrophoresis SDS-PAGE reagent(Jiangsu Biyuntian);pVDF membrane ( Millipore, US); the other reagents are all made in China.

2.3 Laboratory apparatus

Microplate reader (Model:DNM-9602,Beijing Pulang New Technology Co.,Ltd.);Tissue slicing machine (Model:YD-1508B,Zhejiang Jinhua Yidi Medical Equipment Factory);Positive fluorescence microscope FR-4A (Shanghai Optical Instrument Factory); motic3000 microphotographic imaging system(Motic,USA);iCEN-24R desktop high-speed refrigerated centrifuge (Hangzhou Aosheng Instrument Co.,Ltd.);tanon EPS-300 Electrophoresis Instrument (Shanghai Tianneng Technology Co.,Ltd.);Tanon VE-186 electrophoresis tank (Shanghai Tianneng Technology Co.,Ltd.);tanon VE-180B transfer tank (Shanghai Tianneng Technology Co., Ltd.);Tanon-4600 gel imaging system(Shanghai Tianneng Technology Co., Ltd.);image-pro plus6.0 image analysis software.

2.4 Grouping and administration

24 rats were randomly divided into 4 groups (blank group, model group,Yigan capsule low dose group,Yigan capsule high dose group).In addition to the blank group (n = 6), the remaining 18 only with 50 mg/kg INH + 50 mg/kg RFP mixture[8] (INH dissolved in sterile distilled water, RFP dissolved in pH3.0 hydrochloric acid solution),the model was replicated by continuous intragastric administration for 28 d.After the model was successfully reproduced, the lowdose (0.468 g/kg)and high-dose (1.872 g/kg)of Yigan Capsule were continuously intragastrically administered for 4 weeks, and the remaining 2 groups were intragastrically administered with an equal volume of 0.9 % sodium chloride solution.

2.5 Detection indicators and methods

2.5.1 Pathological changes of liver tissue were observed by HE staining

After the last gavage, the rats were fasted but not watered for 12 h.After intraperitoneal injection of 10% chloral hydrate anesthesia,the liver was removed and fixed with 10% neutral formaldehyde for 48 h, then the tissue was dehydrated and transparent, and then embedded in paraffin sections, and finally observed by HE staining;The remaining tissues were stored in a refrigerator at 80 ℃.

2.5.2 Detection of liver function indexes in serum of rats

Appropriate amount of blood was collected by vacuum blood collection tube and placed at 4 ℃ for 12 h, Centrifuge at 3 500 rpm for 10 min and remove supernatant, ALT, AST, ALP, γ-GT and TBIL were detected according to the kit instructions.

2.5.3 The expression of HMGB1, NF-κBp65 and RAGE protein in liver tissue was detected by IHC

The liver tissue paraffin sections prepared under ‘1.5.1’ were used to measure HMGB1, NF-κBp65 and RAGE proteins in the liver by PV two-step method.The sections were dewaxed to water, microwave antigen repair, primary antibody incubation(concentration of 1: 100)4 overnight, after incubation with secondary antibody, color development, re-staining, dehydration and transparent sealing, Motic3000 microscope photography system was used to take photos, and three discontinuous fields were randomly selected for each section, Image-pro plus6.0 image analysis software was used to analyze the positive expression in the selected area.The average integral optical density (IOD)of the positive expression was used to represent the relative expression of the protein.

2.5.4The expression of HMGB1, NF-κBp65, RAGE, TNFand IL-1 in liver tissue was detected by WB

An appropriate amount of liver tissue was added to the RIPA protein lysate, and the sample was obtained by grinding, low-temperature centrifuge centrifugation, supernatant extraction, and metal bath protein denaturation.Then sample loading, electrophoresis, transfer membrane,blocking, primary antibody incubation overnight at 4 ℃(concentration of 1:1 000), washing membrane, secondary antibody incubation (concentration of 1 : 2 000), ECL color development after shooting.The gray values of the target protein bands and the internal reference protein bands in each group were measured by Imagejwin64 software, and then the results of the gray value of the target protein bands were calculated compared with the gray value of the internal reference protein bands, which were used to represent the relative expression of the target protein.

2.6 Statistical method

SPSS 26.0 software was used for statistical analysis.All experimental data were expressed as mean ± standard deviation (±s).The experimental data were analyzed by single factor ANOVA.The t test was used for comparison between groups.P＜0.05 was considered statistically significant.

3.Results

3.1 General observation

During the experiment, the diet and drinking water of the blank rats were normal, their behaviors were active, their movements were flexible, their hair was smooth and neat, and their urine was normal;The rats in the RIF + INH model group showed sluggishness,slow activity, decreased food intake, yellow and dry hair, yellow urine, and different degrees of orange changes in feces ; In contrast,the performance of rats treated with Yigan capsule was significantly better than that of the model group.

3.2 Effect of Yigan Capsule on pathological damage of liver tissue in ATB-DILI rats

Under the microscope, the liver tissue structure of the blank group (A)was complete, the liver plate was radially arranged with the central vein as the center, and there was no inflammatory cell infiltration; Compared with the blank group, the arrangement of liver tissue in the model group (B)was disordered, the number of karyopyknosis increased, the liver cells were swollen and fused,the boundary of cytoplasm and nucleus was not clear, the number of inflammatory cells increased, and the liver tissue was spotted necrosis; Compared with the model group, the liver cell structure of the Yigan capsule treatment group was clear, the hepatic lobules were arranged neatly, the number of nuclear condensation was reduced,and the number of inflammatory cells was reduced, and the effect of the high dose group (D)was more significant than that of the low dose group (C).See Figure 1.

3.3 Effect of Yigan Capsule on liver function indexes in serum of ATB-DILI rats

The contents of ALT, AST, ALP, γ-GT and TBIL in the model group were significantly higher than those in the blank group(P＜0.05); After administration of Yigan capsule, the above liver function indexes in serum of rats were significantly improved(P＜0.05).See table 1.

3.4 The effect of Yigan Capsule on NF-κBp65, RAGE and HMGB1 in liver tissue of ATB-DILI rats

IHC results showed that NF-κBp65, RAGE and HMGB1 proteins were mainly expressed in the cytoplasm of the liver, showing yellow or brownish yellow.Compared with the blank group,the expression of yellow or brown-yellow in the liver tissue of the model group was significantly increased (P＜0.05);The expression of yellow or brown in each treatment group of Yigan capsule was significantly reduced(P＜0.05).See Table 2,Figure2.

3.5 The effect of Yigan Capsule on the expression of TNF-α and IL-1 protein in liver tissue of ATB-DILI rats

WB results showed that compared with the blank group, the expression of TNF-α and IL-1 protein in the liver tissue of the model group increased to varying degrees (P＜0.05); Compared with the model group, the expression of TNF-α and IL-1 protein in each treatment group of Yigan capsule was significantly decreased(P＜0.05).See Table 3, Figure 3.

Fig1 HE staining results of rat liver tissue in each group (×200)

Tab 1 The levels of ALT, AST, ALP, γ-GT and TBIL in serum of rats in each group were compared.(n=6, ±s)

Tab 1 The levels of ALT, AST, ALP, γ-GT and TBIL in serum of rats in each group were compared.(n=6, ±s)

Note: Compared with the blank group: * P ＜ 0.05; compared with the model group: # P ＜ 0.05

Group ALT (U/L) AST (U/L) ALP (U/L) γ-GT (U/L) TBIL (μmol/l)Blank 48.02±4.38 84.07±4.40 180.54±13.24 61.28±4.65 5.07±0.25 Model 76.51±5.28* 142.71±6.38* 308.10±14.21* 107.97±6.53* 13.76±0.77*Low dose 63.15±2.46# 118.95±5.96# 247.93±10.65# 87.22±3.54# 9.58±0.56#High dose 56.27±3.74# 105.30±8.04# 225.21±77.02# 79.52±2.31# 8.15±0.35#

Fig 2 Positive expression of NF-κBp65, RAGE and HMGB1 protein in liver tissue of rats in each group(IHC,×200)

3.6 The effect of Yigan Capsule on the expression of NFκBp65, RAGE and HMGB1 protein in liver tissue of ATBDILI rats

WB results showed that compared with the blank group, the expression of NF-κBp65, RAGE and HMGB1 protein in the liver tissue of the model group was significantly increased (P＜0.05);Compared with the model group, the expression levels of NFκBp65, RAGE and HMGB1 protein in each treatment group of Yigan capsule were significantly decreased (P＜ 0.05).See table 4,figure 3.

Tab 2 Expression of NF-κBp65, RAGE and HMGB1 protein in liver tissue of rats in each group(n=6, ±s)

Tab 2 Expression of NF-κBp65, RAGE and HMGB1 protein in liver tissue of rats in each group(n=6, ±s)

Note: Compared with the blank group: * P ＜ 0.05; compared with the model group: # P ＜ 0.05

Group NF-kBp65 RAGE HMGB1 Blank 97.58±6.27 56.88±2.85 78.32±3.37 Model 162.15±1.49* 225.63±13.96* 171.57±5.69*Low dose 144.43±2.10# 105.95±2.08# 115.25±3.76#High dose 123.69±3.52# 68.52±11.26# 85.39±4.01#

Tab 3 Comparison of TNF-α and IL-1 levels in liver tissue of rats in each group(n=3, ±s)

Tab 3 Comparison of TNF-α and IL-1 levels in liver tissue of rats in each group(n=3, ±s)

Note: Blank group comparison: * P ＜ 0.05; compared with the model group:# P ＜ 0.05

Group TNF-a/b-Actin IL-1b/b-Actin Blank 0.74±0.02 0.68±0.01 Model 1.19±0.04* 1.15±0.02*Low dose 0.87±0.04# 0.85±0.01#High dose 0.79±0.02# 0.81±0.02#

Fig 3 Electrophoretogram of NF-κBp65, RAGE and HMGB1 protein expression in liver tissue of rats in each group

Tab 4 Comparison of NF-κBp65, RAGE and HMGB1 protein expression levels in liver tissue of rats in each group (n=3, ±s)

Tab 4 Comparison of NF-κBp65, RAGE and HMGB1 protein expression levels in liver tissue of rats in each group (n=3, ±s)

Note: Blank group comparison: * P ＜ 0.05; compared with the model group: #P ＜ 0.05

Group NF-kBp65/b-Actin RAGE/b-Actin HMGB1/b-Actin Blank 1.15±0.03 0.81±0.03 0.97±0.02 Model 1.71±0.05* 1.33±0.06* 1.21±0.02*Low dose 1.41±0.04# 0.96±0.01# 1.06±0.01#High dose 1.19±0.01# 0.86±0.02# 0.98±0.01#

4.Discussion

Tuberculosis (TB)is a chronic infectious disease caused by Mycobacterium tuberculosis.According to WHO data, in 2021,the number of new tuberculosis patients worldwide will reach 10.6 million, and the incidence rate will rise by 3.6% compared with 2020[2], It has posed a serious threat to human health.Antituberculosis drugs rifampicin and isoniazid can effectively treat tuberculosis, but long-term use can cause liver cell damage and even lead to acute liver failure.Therefore, it is very important to find a drug treatment that can effectively prevent and treat ATB-DILI [9,10].ALT, AST, ALP, γ-GT and TBIL are still important indicators for the diagnosis and prognosis of ATB-DILI[11].Our experimental results showed that compared with the blank group, the levels of ALT, AST, ALP, γ-GT and TBIL in the serum of the model group were significantly increased, HE pathological results showed that the hepatic lobules were disordered, the hepatocytes were swollen and fused, accompanied by inflammatory cell infiltration and local spotty necrosis, suggesting that the ATB-DILI model was successfully prepared.After the intervention of Yigan Capsule, the levels of the above indicators were significantly reduced, and the pathological damage of the liver was also significantly improved, indicating that Yigan Capsule has a good prevention and treatment effect on ATBDILI.

HMGB1 is a DNA-binding nucleoprotein that is widely present in tissues such as liver, heart, brain, lung and kidney.It can be actively released under cytokine stimulation, or passively released into the extracellular space during cell damage and death[12,13].RAGE is expressed in hepatocytes and macrophages[14] and is a receptor with high affinity for HMGB1.The released extracellular HMGB1 binds to RAGE and activates PI3K/Akt, ERK1/2, p38 MAPK and other pathways, which initiates the downstream nuclear transcription factor(NF-κB)and up-regulates the expression of HMGB1, TNF-α, IL-6 and IL-1β, These inflammatory factors act as agonists of the NFκB signaling pathway, feedback regulation of the NF-κB pathway,induce the waterfall effect, so that inflammation continues to occur,further aggravating inflammatory damage[17,16,15].He Ling et al[18] found that the expression of HMGB1, RAGE and NF-κB in liver tissue of ATB-DILI model rats was significantly increased,suggesting that HMGB1/RAGE signaling pathway may be an important mechanism of ATB-DILI induced by isoniazid combined with rifampicin.The results of this experiment also showed that the expression of HMGB1, RAGE and NF-κBp65 protein in the liver tissue of the model group was significantly increased,which was consistent with the above results, At the same time, the expression of TNF-α and IL-1β protein was also significantly increased, suggesting that HMGB1 combined with RAGE activated the NF-κB signaling pathway in the ATB-DILI model, causing an inflammatory cascade, thereby aggravating the pathological damage of liver tissue.

Salvia miltiorrhiza, Sophora flavescens and Schisandra three drugs together constitute the Yigan capsule.Previous clinical studies have shown that Yigan Capsule has a good effect in the treatment of viral hepatitis, alcoholic liver, fatty liver and other diseases[19].Previous studies in our laboratory have found that Yigan Capsule can inhibit the expression of HMGB1, inflammatory factors TNF-α and IL-6 in liver tissue[7].Studies have shown that cryptotanshinone can inhibit the activation of NF-κB and inhibit the expression of inflammatory factors TNF-α, IL-1β and IL-6[20] ; danshensu can inhibit the transcription and translation of HMGB1 to reduce the synthesis and release of HMGB1, and can also up-regulate TGF-β to reduce the expression of TNF-α and inhibit the release of inflammatory factors[21];Tanshinone IIA has a protective effect on ATB-DILI mice,and its mechanism is related to the activation of SIRTI/NF-κB signaling pathway to reduce the expression of TNF-α and IL-1β[22].Matrine can inhibit the HMGB/TLR4/NF-κB pathway, reduce the inflammatory response, and promote the recovery of gastric ulcer[23].The protective effect of matrine on acetaminophen-induced liver injury is related to inhibiting the expression of inflammatory factors and improving the body ‘s antioxidant capacity[24].Schisandra derivative drugs can protect liver injury by inhibiting the activity of CYP450 enzymes, enhancing antioxidant response and antiinflammatory pathways[25]; Schisandra lignan extract can inhibit the activity of NF-κB and reduce the expression of its downstream inflammatory factors TNF-α and IL-1β, so that the liver tissue of mice is protected from CCL4-induced damage[26].And clinical studies have found that Schisandra can effectively reduce ATBDILI, and all indicators are better than the simple tuberculosis drug treatment group[27].The results of this experiment showed that the expression of HMGB1, RAGE and NF-κBp65 protein in liver tissue of rats in Yigan capsule intervention group was significantly decreased, while the expression of TNF-α and IL-1β protein was also down-regulated.It is suggested that Yigan Capsule can downregulate the protein expression of HMGB1 and RAGE in the liver tissue of ATB-DILI model rats, and further inhibit the expression of NF-κB and its downstream pro-inflammatory factors TNF-α and IL-1β, thereby reducing the pathological damage caused by ATBDILI.

In summary, Yigan Capsule may regulate the expression of NFκB protein through HMGB1/RAGE signaling pathway, inhibit the levels of inflammatory factors such as TNF-a and IL-1β, block the process of inflammatory cascade reaction, and thus play a better role in the prevention and treatment of ATB-DILI.However, the positive control group was not designed in this experiment, which could not form a control relationship with the Yigan capsule treatment group,so that it could not better reflect the effect of Yigan capsule on ATBDILI.

Authors’ contribution

Tang Ya: responsible for the writing of articles, statistical analysis of data; li Jun and Qi Yazhi: responsible for experimental operation and index detection; cao Rui and Zhai Yanling: responsible for the review of relevant literature and the integration of data; han Yusheng:responsible for experimental technical guidance; xu Qiang: put forward the experimental scheme, the article modified.

All authors declare that there is no conflict of interest.