| Objective: Based on Chinese classic theory "liver-spleen concurrent treatment" combined with the modern medical theory of "gut-liver axis" and the previous clinical and experimental studies, this study investigates the effect of HZYGJWF on liver and gut Toll-like receptor signaling pathway in mice with concanavalin A(Con A)-mediated chronic and immunological liver injury so as to clarify the molecular responses in liver damage and intestinal immune barrier and their functions and mechanisms in the initiation and development of infectious diseases, and analyze the key protein targets involved in this process, thereby providing a scientific experimental basis for the clinical application and treatment of liver disease, as well as providing new ideas and new strategies for the prevention and treatment of chronic hepatitis.Methods: Forty mice were randomly divided into four groups, including control group, model group, Diammonium glycyrrhizinza group and HZYGJWF group with 12 in each. 0.2m L 0.9% normal saline was injected by cauda vein in the control group weekly, while Con A 6mg·kg-1 w-1 injected by cauda vein in the remaining three groups weekly.The administration lasted six weeks to build the chronic and immunological liver injury model. After modeling, the control group and the model group were administrated intragastrically with normal saline,the Diammonium glycyrrhizinza group was intragastrically given with 0.68g·kg-1d-1,and the HZYGJWF group intragastrically with 15.17g·kg-1d-1 HZYGJWF;the administration lasted four weeks.The control group and the model group were given distilled water, 9 a.m. and 3p.m. two times daily for 4 weeks. Intragastric administration after the end of the night the day, the morning fasting, collection of mice feces samples, then collected the blood and liver,ileum tissue. The second day early in the morning to collect the feces, blood,liver,ileum specimens of mice.The ALT, AST and TBIL contents in serum were detected using biochemical analysis; the liver tissue was made into pathologic slices to assess liver tissue lesion scores using HE staining; the liver tissue was made into pathologic slices, which was processed with HE staining and then observed under optical microscope for the intestinal mucosa lesions of each group; the ultrastructural pathology of the liver and ileal tissues of each group was observed using electron microscope; the TNF-α, IL-6 and LBP levels in serum were detected using ELASA;the plasma endotoxin levels was determined using tachypleus amebocyte lysate(TAL) chromogenic substrate method;the TLR4,My D88 and NF-k B protein expression levels in mouse liver tissue were detected using Western blot;the TLR4 m RNA,My D88 m RNA,and NF-κB m RNA expression levels in mouse liver tissue were detected using RT-PCR.RT-PCR was used to determine the content of key bacteria in mice’s feces.Results: 1.ALT, AST and TBi L conditions: Compared with the control group, the ALT, AST and TBi L in mouse serum in the model group were significantly increased, suggesting statistically significant differences; compared with the model group, the ALT, AST and TBi L levels in the Diammonium glycyrrhizinza group and the HZYGJWF group showed a downtrend, indicating statistically significant differences; in comparison between the Diammonium glycyrrhizinza group and the HZYGJWF group, the ALT, AST and TBi L in the Diammonium glycyrrhizinza group decreased more, showing statistically significant differences.2.Histopathological changes: under the optical microscope, model group mice liver congestion, portal area with lots of lymphocytes infiltration, fibrous tissue hyperplasia of the liver; compared with the model group, there is statistical significance of HZYGJWF formula group difference in reducing the integral liver lesion(P<0.05). In the liver ultrastructure, model group liver cell nuclear pyknosis, cell membrane morphology is not complete, cytoplasm rarefaction, mitochondria swelling, cristae vague, dilatation of endoplasmic reticulum; HZYGJWF formula group in the above aspects were improved compared with model group. Study showed ileal tissue pathology in mice, the model group of mouse ileum mucosa layer becomes thinner, degeneration of epithelial cells shedding, villous edema, arranged in disorder, lymphocyte infiltration increased; HZYGJWF formula group in the above aspects were lower than model group had significant improvement.3. Expression of TNF-α, IL-6: The TNF-α and IL-6 levels in mouse serum in the model group were significantly higher than the control group, with statistically significant differences(P<0.05); compared with the model group,the TNF-α and IL-6 expression in the Diammonium glycyrrhizinza group was reduced, suggesting statistically significant differences; compared with the model group, the TNF-α, IL-6 levels in serum in the HZYGJWF group decreased, indicating statistically significant differences.4. Endotoxin and LBP levels: Compared with the control group, the LBP and endotoxin levels in mouse plasma in the model group were obviously increased; the comparison between the model group and the Diammonium glycyrrhizinza group showed no statistical significance; Compared with the model group, the LBP and endotoxin levels in the HZYGJWF group were significantly lowered, suggesting statistically significant differences.5. Expression of TLR pathway in the liver: Compared with the control group, the protein expression of TLR4, My D88 and NF-κB as well as their m RNA transcription levels in liver tissue in the model group were significantly increased; compared with the model group, the TLR4 protein expression and m RNA transcription level in the Diammonium glycyrrhizinza group was reduced, while the My D88 and NF-κB protein expression and m RNA transcription levels showed no statistical significance; compared with the model group, the TLR4, My D88 and NF-κB protein expression and m RNA transcription levels in the HZYGJWF group showed a downtrend.6. By the analysis of Real-Time PCR quantitative results can be seen that the intestinal microflora of mice with chronic immune liver injury model of Bifidobacterium, Lactobacillus number compared with the blank control group decreased significantly(P<0.05), while the number of Escherichia coli, Staphylococcus increased(P<0.05).In HZYGJWF gavage intervention, Real-Time PCR detection results compared with the normal group, four for detecting bacteria close to normal,but the difference was statistically significant(P<0.05),significantly improved compared with the model group(P<0.05).And Diammonium glycyrrhizinza Injection on chronic and immunological liver injury in mice intestinal flora weak effect(P>0.05).Conclusions: In this experiment, the animal model of chronic and immunological liver injury was successfully duplicated. HZYGJWF can significantly reduce Con A induced mice hepatocellular injury and decrease the ALT, AST and TBIL contents in plasma, showing a good effect in protecting liver and lowering transaminase. Pathologically, HZYGJWF can significantly improve the pathological changes in liver tissue and lower hepatocellular injury, indicating a satisfactory role in improving the liver tissue inflammatory symptoms of transgenic mice. According to the exploration on HBV transgenic mice in terms of general state, viral replication level, serum enzymes, pathology, cytokine levels, protein expression and gene transcription, it is believed that the mechanism of HZYGJWF adhering to the "liver-spleen concurrent treatment" in treatment of chronic and immunological liver injury may be as follows:1. Through reducing the TNF-α and IL-6 levels in mice with liver injury, decreasing the overstimulation of pro-inflammatory cytokines and inhibiting the expression of TLR4, HZYGJWF can suppress NF-κB activation, regulate TLR4-My D88-NF-κB signaling pathway, and eventually alleviate body’s inflammatory response. The expression changes and interaction of these proteins and cytokines constitute the biological basis of "liver-spleen concurrent treatment" and serve as the targets of HZYGJWF’s immune regulation function.2. HZYGJWF can reduce LPS level, which contributes to the improvement of the intestinal permeability in chronic liver injury mice, lower the levels of endotoxin and its ligand LBP in mouse plasma, and have an obvious blocking effect on endotoxin biological effects. Through protecting the intestinal mucosa, reducing the generation of endotoxin, adjusting "gut-liver axis" status and regulating body’s immune response, HZYGJWF plays a hepatoprotective effect.HZYGJWF can improve intestinal pathological damage, protect intestinal mucosal barrier function, regulate intestinal flora, and reduce the endotoxin produced by opportunistic pathogens. So it can relieve the chronic autoimmune liver injury to a certain extent. And it is proved that Gut-liver Axis has played an important role in pathogenesis of liver injury. |
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