Protection effect of fire capsule on D-GalN / LPS (D-GalN / Lipopolysaccharide)-induced acute hepatic failure mouse

Abstract

The invention relates to the field of medicine application, in particular to application of a fire capsule to treatment and / or prevention of hepatic failure. The fire capsule disclosed by the invention has a good prevention and treatment effect on mouse hepatic failure caused by D-GalN / LPS, can obviously improve the liver function level, has a regulation and control effect on a plurality of inflammation-related signal channels, enhances the oxidative stress resistance of a body, inhibits liver cell apoptosis, obviously relieves hepatic tissue pathological injury caused by D-GalN / LPS, and has an obvious liver protection effect.

Description

Technical Field

[0001] The present invention relates to the field of drug application, in particular to the protective effect of Huoqian capsule on mice with acute liver failure induced by D-GalN / LPS. Background Art

[0002] Acute liver failure (ALF) is a severe liver syndrome characterized by a rapid decline in liver function and diffuse hepatocellular necrosis, triggered by multiple factors. Complications often include coagulation disorders, electrolyte imbalances, and cognitive impairment. The disease progresses rapidly, with a high short-term mortality rate. Currently, no specific medication exists. Acute liver failure falls under the umbrella of Zhuang medicine's "Mi Die Gu" (Mi Die Gu) concept, which holds that "poison and deficiency lead to all diseases." There is no specific treatment for ALF, and liver transplantation is the only effective treatment. This suggests that the toxicity in liver failure is primarily due to the accumulation of inflammatory factors, which exceeds the liver's metabolic and excretion capacity. Combined with decreased liver function (i.e., deficiency), these malignant factors can lead to reversible or irreversible liver damage. Zhuang medicine believes that this is caused by poison accumulating in the liver, overexerting qi and yin, leading to qi and yin deficiency, stagnation (i.e., qi and blood stasis), dysfunctional drainage, and liver dysfunction. Treatment should focus on "regulating qi, replenishing deficiency, and removing toxins," with removing toxins being the primary priority in treating "Gu."

[0003] Zhuang medicine prescriptions are a treasure trove of traditional Chinese medicine resources with distinct ethnic and regional characteristics. They represent the valuable experience of ethnic minorities in their long struggles against disease, passed down through generations of folk pharmacists and boast proven efficacy. Huoqian Capsule, a Zhuang medicine formula derived from proven folk prescriptions and formulated as a preparation, has been designated a key Guangxi Science and Technology R&D project. This prescription, composed of eight Zhuang herbs—Panax notoginseng, Lespedeza serrata, Cyperus rotundus, Plowhead herb, Bat grass, Marchantia striata, Cyclocarya paliurus, and Firewood, has the efficacy of detoxification, swelling reduction, clearing heat and removing dampness. It is primarily used as an adjunctive treatment for scrofula, furuncles, carbuncles, goiters, and acute and chronic hepatitis, cirrhosis, and early-stage liver cancer. Modern research has shown that all six herbs in Huoqian Capsule—Panax notoginseng, Plowhead herb, Bat grass, Marchantia striata, Cyclocarya paliurus, and Firewood—have anti-hepatic activity. This study used a D-galactosamine (D-GalN) / lipopolysaccharide (LPS)-induced acute liver failure model in mice. By observing the effects of Huoqian Capsule on liver tissue damage and LPS-related signaling pathway protein expression in mice with liver failure, it provides experimental and theoretical basis for the in-depth research and clinical application of Huoqian Capsule. Summary of the Invention

[0004] In order to overcome the shortcomings of the existing technology, the present invention provides a method for treating liver failure in mice induced by D-GalN / LPS by using Huoqian Capsule, which can significantly improve liver function levels, regulate multiple inflammation-related signaling pathways, enhance the body's ability to resist oxidative stress, inhibit hepatocyte apoptosis, and significantly reduce liver tissue pathological damage caused by D-GalN / LPS, thereby having a significant liver-protective effect.

[0005] The Huoqian Capsule described in the present invention is a Zhuang medicine formula derived from a folk prescription and prepared as a preparation. It has been listed as a key scientific and technological research and development project in Guangxi. The formula consists of eight Zhuang medicines: Gynura divaricata (L.) DC., Lespedeza formosa (Vog.) Koehne, Tetracera asiatica (Lour.) Hoogland, Viola inconspicua Blume, Christia vespertilionis (Lf) Bahn.f., Marchantia convoluta Gao et Chang., Cylocarya paliurus (Batal.) lljinskaja, and Polygonum chinense L.

[0006] The present invention is achieved through the following technical solutions: (1) Animal model establishment: Mice in the normal group and model group were orally administered with 0.5% CMC-Na at 20 mL / kg, while mice in the other groups were orally administered with silybin or different concentrations of Huoqian capsule at 20 mL / kg, once a day for 10 consecutive days. After fasting for 14-16 h, 1 h after the last dose, mice in the normal group were intraperitoneally injected with 10 mL / kg of normal saline, while mice in the other groups were intraperitoneally injected with 10 mL / kg of a mixture of D-GalN and LPS (D-GalN, 700 mg / kg; LPS, 10 μg / kg).

[0007] (2) Detection of liver function and inflammatory factors: 6 h after modeling, 3 mL of blood was collected from the retroorbital venous plexus of the first 10 surviving mice in each group. After standing for 1–2 h, the blood was centrifuged at 3000 rpm for 10 min, and the supernatant was collected for the detection of ALT and AST liver function indicators and IL-1β, IL-6, IL-10, TNF-α, and MCP-1 inflammatory factors.

[0008] (3) Organ index test of experimental mice: After blood collection, the mice were killed, the livers were quickly separated and weighed, and the organ coefficient (g / 100g) was calculated. Organ index = 100 × (organ weight / body weight).

[0009] (4) Pathological examination of experimental mice: Changes in liver tissue color, texture, size, etc. were observed, recorded, and photographed. The left upper lobe of the liver was isolated and fixed in 10% formalin solution. Fresh 10% formalin solution was replaced once after 6 hours. After fixation for 24 hours, pathological slides were prepared and stained with hematoxylin-eosin (HE). Histopathological changes were observed under a light microscope.

[0010] (5) The left lower lobe of the liver was isolated. Approximately 300 mg of liver tissue was collected from the same site and added to ice-cold physiological saline at a liver weight:volume ratio of 1:9. The liver tissue was minced and made into a 10% liver tissue homogenate using a homogenizer. The mixture was centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The protein level (μg / mL) in the liver tissue homogenate supernatant was determined using the BCA assay.

[0011] (6) Detection of oxidative stress indicators and inflammatory factor indicators: The left lower lobe of the liver was quickly separated, and about 200 mg of liver tissue was collected from the same part. Ice-cold physiological saline was added at a liver weight: volume ratio of 1:9, and the liver was minced. A 10% liver tissue homogenate was prepared using a homogenizer, and centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and used for the detection of oxidative stress indicators such as SOD, MDA, and NO levels, and the detection of inflammatory factor indicators such as IL-1β, IL-6, and TNF-α. (7) Take about 20 mg of mouse liver tissue, add 350 μL of lysis buffer LB, use a high-speed low-temperature tissue grinder to homogenize, repeatedly use DNA removal / RNA adsorption universal columns, purify 7 times, obtain RNA solution, use a thermal cycler to treat for 2 minutes at 42°C to remove genomic DNA, and reverse transcribe the reaction system on a thermal cycler at 37°C for 15 minutes, 85°C for 5 seconds to obtain cDNA. Operate 20 μL of the amplification system according to the instructions and perform the reaction on a real-time fluorescence quantitative RT-PCR instrument. The PCR amplification conditions are 95°C pre-denaturation for 30 seconds, denaturation for 5 seconds, 55°C annealing for 30 seconds, and 72°C full extension for 30 seconds, for a total of 45 cycles. β-actin was used as an internal reference and 2 -ΔΔCT The relative expression levels of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α mRNA were calculated by the method. (8) Approximately 20 mg of mouse liver tissue was placed in a 2 mL grinding tube and RIPA lysis buffer was added at a ratio of 1:10. The tissue was ground four times in a high-speed low-temperature tissue grinder at -20°C, refrigerated for 30 min, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The protein concentration was determined by the BCA method. An appropriate amount of SDS-PAGE protein loading buffer was added to the supernatant, mixed thoroughly, and denatured at 95°C in a thermal cycler for 15 min. The cells were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was placed in an incubation box and blocked with 5% skim milk for 2 h. The PVDF membrane was incubated with the primary antibodies Bax (1:2000), Bcl-2 (1:1000), Bcl-xl (1:2000), p38 (1:1000), p-p38 (1:2000), p65 (1:2000), p-p65 (1:2000), and β-actin (1:50000) on a shaker overnight at 4°C. The PVDF membrane was washed three times with TBST at room temperature in the dark for 5 min each time. The secondary antibody (goat anti-rabbit 1:5000) was incubated on a shaker at room temperature for 2 h. The PVDF membrane was washed three times with TBST at room temperature in the dark for 10 min each time. ECL chemiluminescence development and fixation were performed, and the bands were exposed using the Tanon fluorescence image analysis system and scanned using Gel-Proanalyzer 4 software to obtain protein integrated optical density (IOD) values. The expression levels of Bax, Bcl-2, Bcl-xl, p38, p-p38, p65, and p-p65 proteins were analyzed.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. The Huoqian Capsule provided by the present invention has an effect on acute liver failure induced by D-GalN / LPS. Among the liver function indicators tested, the ALT and AST levels of mice receiving the Huoqian Capsule were significantly reduced (P < 0.01), and it has a significant improvement effect on the abnormal liver function indicators of mice caused by D-GalN / LPS.

[0013] 2. The Huoqian capsule provided by the present invention significantly reduced the levels of IL-1β, IL-6, TNF-α, and MCP-1 in the serum of mice (P < 0.05 or P < 0.01), and significantly increased the level of IL-10 (P < 0.05 or P < 0.01). It also significantly improved the abnormal serum inflammatory factor indicators of mice induced by D-GalN / LPS.

[0014] 3. The Huoqian capsule provided by the present invention significantly reduced the MDA and NO levels in the liver tissue of mice (P < 0.05 or P < 0.01), and significantly increased the SOD level (P < 0.05 or P < 0.01), and had a significant improvement effect on the abnormal oxidative stress indicators in the liver tissue of mice induced by D-GalN / LPS.

[0015] 4. The Huoqian capsule provided by the present invention significantly reduced the levels of IL-1β, IL-6 and TNF-α in the liver tissue of mice (P < 0.05 or P < 0.01), and had a significant improvement effect on the abnormal inflammatory factor indicators in the liver tissue of mice induced by D-GalN / LPS.

[0016] 5. The Huoqian Capsule provided by the present invention can alleviate pathological damage to liver tissue in mice. The area of ​​necrosis is significantly reduced, the outline of hepatocyte structure is clearly defined, the arrangement of hepatic cords is clearly orderly, and the arrangement of hepatocytes is clearly regular. In the model group, the outline of hepatocyte structure is blurred, the arrangement of hepatic cords is disordered, and the hepatocytes show diffuse necrosis. Numerous red blood cells are observed intercellularly, and the cell nuclei show karyopyknosis or effacement. It can significantly alleviate the pathological damage of liver tissue caused by D-GalN / LPS.

[0017] 6. The Huoqian capsule provided by the present invention showed that the mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6, and TNF-α in the liver tissue of mice were all downregulated, with the mRNA expressions of NF-κB, IκBα, TLR4, IL-1β, and TNF-α in the medium and high dose groups being significantly downregulated (P < 0.05 or P < 0.01), and the mRNA expressions of iNOS and IL-6 in the high dose group being significantly downregulated (P < 0.05 or P < 0.01). The Huoqian capsule can significantly improve the mRNA expression levels of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6, and TNF-α in the inflammatory signaling pathways induced by D-GalN / LPS.

[0018] 7. Huoqian Capsules provided by the present invention significantly downregulated Bax protein expression in mouse liver tissue (P < 0.05 or P < 0.01), significantly upregulated Bcl-2 protein expression (P < 0.01), and upregulated Bcl-xl protein expression, with the difference in the high-dose group being significant (P < 0.01). They also significantly inhibited D-GalN / LPS-induced hepatocyte apoptosis. 8. The Huoqian capsule provided by the present invention significantly downregulated the expression of p-p38 and p-p65 in the liver tissue of mice (P < 0.05 or P < 0.01), and significantly improved the expression level of p38MAPK / NF-κB protein in the D-GalN / LPS-induced liver inflammation signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HE staining was used to observe the pathological changes of liver tissue in mice with acute liver failure induced by D-GalN / LPS after Huoqian Capsule was applied.

[0020] Figure 2 This is the effect of Huoqian Capsule on the expression of Bax, Bcl-2 and Bcl-x1 proteins in liver tissue of mice with acute liver failure induced by D-GalN / LPS.

[0021] Figure 3 This is the effect of Huoqian Capsule on the expression of p-p38 and p-p65 proteins in liver tissue of mice with acute liver failure induced by D-GalN / LPS. DETAILED DESCRIPTION

[0022] Example 1 Establishment of D-GalN / LPS-induced acute liver failure model. experimental animals Animals were provided by Hunan Slake Jingda Laboratory Animal Co., Ltd., with production license number SCXK(Xiang)2019-0004 and laboratory animal quality certificate number 430727231101386956. This study was reviewed and approved by the Ethics Committee of Guangxi University of Chinese Medicine, in accordance with the 3R principles, with approval number DW20230620-129.

[0023] Reagents Huoqian capsule (Guangxi Baicaotang Pharmaceutical Co., Ltd., 20230513011); silybin (Shanghai McLean Biochemical Technology Co., Ltd., C14554655); sodium carboxymethyl cellulose (CMC-Na, Shanghai McLean Biochemical Technology Co., Ltd., C14834490); D-GalN (Shandong Xiya Chemical Co., Ltd., 20230420); LPS (Beijing Solaibao Technology Co., Ltd., 1031Q0312); alanine aminotransferase (ALT), aspartate aminotransferase (AST), protein quantitative BCA assay, nitric oxide (NO), superoxide dismutase (SOD), and malondialdehyde (MDA) assay kits (Nanjing Jiancheng Bioengineering Institute, batch no. 20230914, 20230923, 20231016, 20231013, 20230925, and 20231013); interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 (IL-10) ELISA kits (Jiangsu Enzyme Immunity Industry Co., Ltd., batch number 202309); RIPA cell lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd., P0013); SDS-PAGE protein loading buffer (5×) (Hefei White Shark Biotechnology Co., Ltd., BL502A); Immobilon-PSQ PVDF membrane (Sigma-Aldrich, USA, ISEQ00010); skim milk powder (Wuhan Sevier Biotechnology Co., Ltd., GC310001); ECL luminescent solution (Suzhou Sizhengbai Biotechnology Co., Ltd., 17046); goat anti-rabbit secondary antibody (Changzhou Xiangtai Biotechnology Co., Ltd., S0001); primary antibodies against β-actin, Bax, and Bcl-2 (Wuhan Aibote Biotechnology Co., Ltd., AC026, A19684, and AC026); primary antibodies against Bcl-xl, p-p38, and p65 (Beijing Aibote Biotechnology Co., Ltd., 10783-1-AP, 28796-1-AP, and 80979-1-RR).

[0024] instrument QuantStudio TM3 real-time fluorescence quantitative RT-PCR instrument, TCA0096 thermal cycler (ThermoFisher, USA); KZ-III-F high-speed cryogenic tissue grinder, DS-H200 digital horizontal shaker (Wuhan Sevier Biotechnology Co., Ltd.); H2050R low-temperature centrifuge (Hunan Xiangyi Experimental Instrument Factory); XH-B vortex mixer (Jiangsu Kangjian Medical Products Co., Ltd.); ES2220M electronic balance (Precisa, Switzerland); 5810R high-speed refrigerated centrifuge (Eppendorf, Germany); Epoch2 full-wavelength microplate reader (Bio-Tek, USA); MDF-U74V ultra-low temperature refrigerator (Panasonic, Japan); Direct-Q 5UV-R ultrapure water analyzer (Meck Millipore, USA); ECLIPSE Ni-U advanced fluorescence biological upright microscope (Nikon, Japan); JY-SCZ4+ vertical electrophoresis tank (Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.); 5200Multi fluorescence image analysis system (Shanghai Tianneng Technology Co., Ltd.).

[0025] breeding environment Mice were maintained in a barrier environment at the Experimental Animal Center of Guangxi University of Chinese Medicine, meeting SPF requirements. The experimental animal use license number is SYXK(Gui)2019-0001. Central air conditioning was used, with room temperature maintained at 20.0°C–25.0°C, relative humidity maintained at 40.0%–70.0%, and lighting maintained at a 12-hour interval. Mice were housed in non-toxic, high-pressure, high-temperature, and corrosion-resistant IVC plastic cages, with no more than six mice per cage. They were fed an SPF-grade mouse maintenance diet and had free access to food and purified water.

[0026] method One hundred and fifty SPF male KM mice were randomly divided into six groups (n=25) using Excel block-based stratified randomization (Block Groups) based on body weight: a normal group, a model group, a silibinin group (100 mg / kg), and low-, medium-, and high-dose Huoqian Capsules groups (365, 730, and 1460 mg / kg). Mice in the normal and model groups were orally administered 0.5% CMC-Na at 20 mL / kg, while mice in the other groups were orally administered silibinin or different concentrations of Huoqian Capsules at 20 mL / kg once daily for 10 days. After fasting for 14-16 hours, mice in the normal group received an intraperitoneal injection of 10 mL / kg of normal saline, while mice in the other groups received an intraperitoneal injection of 10 mL / kg of a mixture of D-GalN and LPS (700 mg / kg D-GalN and 10 μg / kg LPS).

[0027] Observation of mouse survival rate: After each group is numbered, 15 mice are retained for observation. After modeling, the number of surviving animals in each group is recorded every hour for 8 hours. After 12 hours, the number of surviving animals in each group is recorded every 12 hours. The survival rate of mice is calculated after 72 hours of observation. Survival rate (%) = 100% × (number of surviving animals at 72 hours / number of observed animals)

[0028] Blood collection The first 10 surviving mice in each group were numbered and blood was collected from the retro-orbital venous plexus 6 hours after modeling. After standing for 1 to 2 hours, the blood was centrifuged at 3000 r·min-1 for 10 minutes, and the supernatant was collected for the detection of liver function and inflammatory factor indicators. After blood collection, the mice were killed, the livers were quickly removed, the liver mass was weighed, and the organ index was calculated; the liver tissues of each group of mice were collected from the same part, weighed, and homogenized at a ratio of 1:9 between the liver mass and ice saline, and centrifuged at 3000 r·min-1 for 10 min to obtain the supernatant. The protein level was detected by the BCA method for the detection of oxidative stress indicators and inflammatory factor indicators.

[0029] Sampling and pathological examination After blood collection, the mice were killed, the livers were quickly separated and weighed, and the organ coefficient (g / 100g) was calculated. Organ coefficient = 100 × (organ weight / body weight).

[0030] The left upper lobe of the liver was separated and fixed in 10% formalin solution. Fresh 10% formalin solution was replaced once after 6 hours. After fixation for 24 hours, the samples were dehydrated, embedded, sectioned, spread and dewaxed, and stained with hematoxylin-eosin (HE staining). Changes in the pathological structure of the liver tissue were observed under a light microscope.

[0031] The left lower lobe of the liver was isolated. Approximately 300 mg of liver tissue was collected from the same site. Ice-cold saline was added at a liver weight:volume ratio of 1:9, and the tissue was minced. A 10% liver tissue homogenate was prepared using a homogenizer and centrifuged at 3000 rpm for 10 minutes. The supernatant was collected. Protein levels (μg / mL) in the liver tissue homogenate supernatant were determined using the BCA assay, following the kit instructions.

[0032] Liver function indicators Serum ALT and AST levels were detected using an automatic biochemical analyzer.

[0033] Oxidative stress indicators The supernatant of liver tissue homogenate was collected and its SOD, MDA and NO levels were detected.

[0034] Inflammatory cytokine indicators Serum samples or liver tissue homogenate supernatant were collected and the levels of IL-1β, IL-6, IL-10, TNF-α, and MCP-1 were detected by ELISA.

[0035] About 20 mg of mouse liver tissue was collected, and real-time fluorescence quantitative PCR (RT-PCR) was used to detect the mRNA expression levels of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in liver tissue.

[0036] About 20 mg of mouse liver tissue was collected, and the expression levels of apoptosis and inflammation-related proteins in liver tissue were detected by Western blot.

[0037] Statistics SPSS 17.0 was used for statistical analysis and the data were expressed as (±s). When P < 0.05, the data differences were considered statistically significant.

[0038] The measurement data were normal and homogeneous, and were analyzed by one-way ANOVA test, and the LSD test was used for pairwise comparison analysis. If the data were non-normal or heterogeneous, the Kruskal-Wallis H test was used for analysis, and the Mann-Whitney U test was used for pairwise comparison analysis.

[0039] Example 2 Pharmacological efficacy test of Huoqian capsule against acute liver failure Survival rate Compared with the normal group, the survival rate of mice in the model group was significantly decreased (P < 0.01). Compared with the model group, the survival rates of mice in the silibinin group and the low-, medium-, and high-dose Huoqian Capsule groups were significantly increased (P < 0.01). Therefore, it is believed that D-GalN / LPS can cause a decrease in mouse survival rate, and Huoqian Capsule has a significant effect on improving the D-GalN / LPS-induced decrease in mouse survival rate. See Table 1 for details.

[0040] Table 1 Effect of Huoqian Capsule on the survival rate of mice Tab.1 Effects of Huoqian capsule on mice survival rate Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0041] Liver function indicators Compared with the normal group, the liver index and serum ALT and AST levels of mice in the model group were significantly increased (P < 0.01). Compared with the model group, the liver index and serum ALT and AST levels of mice in the silibinin group and the low-, medium-, and high-dose Huoqian Capsule groups were significantly decreased (P < 0.01). Therefore, it is suggested that D-GalN / LPS can cause abnormal changes in liver function in mice, and Huoqian Capsule has a significant ameliorative effect on these abnormal changes in liver function in mice induced by D-GalN / LPS. See Table 2 for details.

[0042] Table 2 Effects of Huoqian Capsule on liver index and serum ALT and AST levels in mice Tab.2 Effects of Huoqian capsule on liver index and levels of ALT, ASTin mice serum Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0043] Serum inflammatory cytokine indicators Compared with the normal group, the model group showed significantly increased serum IL-1β, IL-6, TNF-α, and MCP-1 levels (P < 0.01), while significantly decreased IL-10 levels (P < 0.01). Compared with the model group, the silibinin group and the low-, medium-, and high-dose Huoqian Capsule groups showed significantly decreased serum IL-1β, IL-6, TNF-α, and MCP-1 levels (P < 0.05 or P < 0.01), while significantly increased IL-10 levels (P < 0.05 or P < 0.01). Therefore, it is suggested that D-GalN / LPS can cause abnormalities in serum inflammatory cytokine indices in mice, and Huoqian Capsule has a significant ameliorative effect on these abnormalities. See Table 3 for details.

[0044] Table 3 Effects of Huoqian Capsule on serum IL-1β, IL-6, TNF-α, IL-10 and MCP-1 levels in mice Tab.3 Effects of Huoqian capsule on levels of IL-1β, IL-6, TNF-α, IL-10, and MCP-1 in mice serum Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0045] Compared with the normal group, the model group showed significantly increased MDA and NO levels in liver tissue (P < 0.01), and significantly decreased SOD levels (P < 0.01). Compared with the model group, the silybin group and the Huoqian Capsule low-, medium-, and high-dose groups showed significantly decreased MDA and NO levels in liver tissue (P < 0.05 or P < 0.01), and significantly increased SOD levels (P < 0.05 or P < 0.01). Therefore, it is believed that D-GalN / LPS can cause abnormalities in oxidative stress indicators in mouse liver tissue, and Huoqian Capsule has a significant ameliorative effect on these abnormalities. See Table 4 for details.

[0046] Table 4 Effects of Huoqian Capsule on SOD, MDA and NO levels in mouse liver tissue Tab.4 Effects of Huoqian capsule on levels of SOD, MDA, and NO in miceliver tissue Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0047] Compared with the normal group, the levels of IL-1β, IL-6, and TNF-α in the liver tissue of mice in the model group were significantly increased (P < 0.01). Compared with the model group, the levels of IL-1β, IL-6, and TNF-α in the liver tissue of mice in the silibinin group and the low-, medium-, and high-dose Huoqian Capsule groups were significantly decreased (P < 0.05 or P < 0.01). Therefore, it is believed that D-GalN / LPS can cause abnormalities in the liver tissue inflammatory cytokine indicators of mice, and Huoqian Capsule has a significant improvement effect on the abnormalities in the liver tissue inflammatory cytokine indicators of mice caused by D-GalN / LPS. See Table 5 for details.

[0048] Table 5 Effects of Huoqian Capsule on IL-1β, IL-6 and TNF-α levels in mouse liver tissue Tab.5 Effects of Huoqian capsule on levels of IL-1β, IL-6, and TNF-α inmice liver tissue Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0049] Pathological microscopic observations revealed that the hepatocytes in the normal group had clear outlines, a purple-red color, and a regular, orderly arrangement. Hepatocyte cords were arranged radially, and the nuclei were large and round, located in the center of the cells. In the model group, the hepatocytes had blurred outlines, disordered hepatocyte cords, and diffuse necrosis. Numerous red blood cells were observed between cells, and the nuclei were karyopyknotic or elusive. Compared with the model group, the silybin group and the low-, medium-, and high-dose Huoqian Capsule groups showed significantly reduced liver tissue pathological damage, a significantly smaller area of ​​necrosis, and clearer hepatocyte outlines, orderly hepatocyte cords, and regular hepatocyte arrangement. This suggests that Huoqian Capsule can alleviate the pathological changes induced by D-GalN / LPS.

[0050] Expression levels of inflammatory signaling pathways Effects on the mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in the liver tissues of mice Compared with the normal group, the mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in the liver tissues of mice in the model group were significantly up-regulated (P<0.01); compared with the model group, the mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in the liver tissues of mice in the silibinin group were significantly up-regulated (P<0.01). NA expression was significantly downregulated (P < 0.01). The mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6, and TNF-α in the liver tissues of mice in the low-, medium-, and high-dose Huoqian Capsule groups were all downregulated. Specifically, the mRNA expressions of NF-κB, IκBα, TLR4, IL-1β, and TNF-α were significantly downregulated in the medium- and high-dose groups (P < 0.05 or P < 0.01), while the mRNA expressions of iNOS and IL-6 were significantly downregulated in the high-dose group (P < 0.05 or P < 0.01). Therefore, it is believed that D-GalN / LPS can lead to abnormal expression of inflammatory signaling pathways in mouse liver tissue, and Huoqian Capsule has a significant ameliorative effect on D-GalN / LPS-induced inflammatory signaling pathways in mouse liver tissue. See Tables 6 and 7 for details.

[0051] Table 6 Effects of Huoqian Capsule on the expression of NF-κB, IκBα, TLR4 and iNOS mRNA in mouse liver tissue Tab.6 Effects of Huoqian capsule on expression of NF-κB, IκB α, TLR4, and iNOS mRNA in mice liver tissue Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01. Table 7 Effects of Huoqian Capsule on the mRNA expression of IL-1β, IL-6 and TNF-α in mouse liver tissue Tab.7 Effects of Huoqian capsule on mRNA expression ofIL-1β, IL-6, and TNF-α in mice liver tissue Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0052] Protein expression level Effects on the expression of apoptosis factors Bax, Bcl-2 and Bcl-xl proteins in the liver tissue of mice Compared with the normal group, the expression of Bax protein in the liver tissue of mice in the model group was significantly upregulated (P < 0.01), and the expression of Bcl-2 and Bcl-xl proteins was significantly downregulated (P < 0.01); Compared with the model group, the expression of Bax protein in the liver tissue of mice in the silybin group was significantly downregulated (P < 0.01), and the expression of Bcl-2 and Bcl-xl proteins was significantly upregulated (P < 0.01); Huoqian Capsule Bax protein expression in the liver tissues of mice in the low-, medium-, and high-dose groups was significantly downregulated (P < 0.05 or P < 0.01), Bcl-2 protein expression was significantly upregulated (P < 0.01), and Bcl-xl protein expression was upregulated. The difference in the high-dose group was significant (P < 0.01). Therefore, it is believed that D-GalN / LPS can cause abnormal protein expression levels in mouse liver tissue, and Huoqian Capsule has a significant effect on improving the protein expression levels in mouse liver tissue caused by D-GalN / LPS. See Table 8 for details.

[0053] Table 8 Effects of Huoqian Capsule on the expression of Bax, Bcl-2 and Bcl-xl in mouse liver tissue Table 8 Effects of Huoqian capsule on Bax, Bcl-2, and Bcl-xl expression in mice liver tissue Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

[0054] Inflammatory signaling pathway protein expression levels Compared with the normal group, the expression of p-p38 and p-p65 in the liver tissue of mice in the model group was significantly upregulated (P < 0.01). Compared with the model group, the expression of p-p38 and p-p65 in the liver tissue of mice in the silibinin group and the low-, medium-, and high-dose Huoqian Capsule groups was significantly downregulated (P < 0.05 or P < 0.01). Therefore, it is believed that D-GalN / LPS can lead to abnormal expression levels of signaling pathway proteins in mouse liver tissue, and Huoqian Capsule has a significant effect on improving the expression levels of protein in the D-GalN / LPS-induced inflammatory signaling pathway in mouse liver tissue. See Table 9 for details.

[0055] Table 9 Effects of Huoqian Capsule on the expression of Bax, Bcl-2 and Bcl-xl in mouse liver tissue Table 9 Effects of Huoqian capsule on Bax, Bcl-2, and Bcl-xl expression in mice liver tissue Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.01.

Claims

1. The protective effect of Huoqian Capsule on mice with acute liver failure induced by D-GalN / LPS.

2. The use according to claim 1, characterized in that Compared with the model group, the liver index and serum ALT and AST levels of mice in the Huoqian capsule groups at all doses were significantly decreased (P < 0.01); compared with the normal group, the liver index and serum ALT and AST levels of mice in the model group were significantly increased (P < 0.01).

3. The use according to claim 1, characterized in that Compared with the model group, the serum IL-1β, IL-6, TNF-α and MCP-1 levels of mice in all doses of Huoqian Capsule groups were significantly decreased (P < 0.05 or P < 0.01), and the IL-10 level was significantly increased (P < 0.05 or P < 0.01); compared with the normal group, the serum IL-1β, IL-6, TNF-α and MCP-1 levels of mice in the model group were significantly increased (P < 0.01), and the IL-10 level was significantly decreased (P < 0.01).

4. The use according to claim 1, characterized in that Compared with the model group, the levels of MDA and NO in the liver tissues of mice in the Huoqian capsule groups at all doses were significantly decreased (P < 0.05 or P < 0.01), and the level of SOD was significantly increased (P < 0.05 or P < 0.01). Compared with the normal group, the levels of MDA and NO in the liver tissue of mice in the model group were significantly increased (P<0.01), and the level of SOD was significantly decreased (P<0.01).

5. The use according to claim 1, characterized in that Compared with the model group, the levels of IL-1β, IL-6 and TNF-α in the liver tissue of mice in all doses of Huoqian Capsule groups were significantly decreased (P < 0.05 or P < 0.01); compared with the normal group, the levels of IL-1β, IL-6 and TNF-α in the liver tissue of mice in the model group were significantly increased (P < 0.01).

6. The use according to claim 1, characterized in that compared with the model group The pathological damage of liver tissue in mice in all Huoqian capsule groups was significantly alleviated, the necrotic area was significantly reduced, the outline of liver cell structure was significantly clearer, the arrangement of hepatic cords was significantly orderly, and the arrangement of liver cells was significantly regular; the outline of liver cell structure in mice in the model group was blurred, the arrangement of hepatic cords was disordered, the liver cells showed diffuse necrosis, a large number of red blood cells were seen distributed between cells, and the cell nuclei showed nuclear condensation or disappeared.

7. The use according to claim 1, compared with the model group, the mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in the liver tissue of mice in each dose group of Huoqian Capsule were downregulated, among which the mRNA expressions of NF-κB, IκBα, TLR4, IL-1β and TNF-α in the medium and high dose groups were significantly downregulated (P < 0.05 or P < 0.01), and the mRNA expressions of iNOS and IL-6 in the high dose group were significantly downregulated (P < 0.05 or P < 0.01); compared with the normal group, the mRNA expressions of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in the liver tissue of mice in the model group were significantly upregulated (P < 0.01). It can significantly improve the mRNA expression levels of NF-κB, IκBα, TLR4, iNOS, IL-1β, IL-6 and TNF-α in the inflammatory signaling pathways induced by D-GalN / LPS.

8. The use according to claim 1, compared with the model group, the expression of Bax protein in the liver tissue of mice in the low-, medium- and high-dose Huoqian Capsule groups were significantly downregulated (P < 0.05 or P < 0.01), the expression of Bcl-2 protein was significantly upregulated (P < 0.01), and the expression of Bcl-x1 protein was upregulated, among which the difference in the high-dose group was significant (P < 0.01); compared with the normal group, the expression of Bax protein in the liver tissue of mice in the model group was significantly upregulated (P < 0.01), and the expression of Bcl-2 and Bcl-xl proteins were significantly downregulated (P < 0.01).

9. The use according to claim 1: Compared with the model group, the expression of p-p38 and p-p65 in the liver tissue of mice in all dose groups of Huoqian Capsule was significantly downregulated (P < 0.05 or P < 0.01); compared with the normal group, the expression of p-p38 and p-p65 in the liver tissue of mice in the model group was significantly upregulated (P < 0.01). The drug can significantly improve the expression level of p38MAPK / NF-κB protein in the D-GalN / LPS-induced liver inflammation signaling pathway.

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