| Purpose:Controlling Chronic hepatitis B has made certain progress in China, but the reality is not optimistic,10%-20%of CHB will develope to liver cirrhosis and1%-5%CHB will develope to primary hepatocellular carcinoma (HCC), so the prevention and controlling situation Chronic hepatitis B is still grim and the chronic hepatitis B viral hepatitis is still a prominent public health problem in China. In the treatment of Chronic hepatitis B interferon and nucleoside analogues is undoubtedly the first choice, but basically the side effects of interferon is difficult to adapt and needs of lifetime use nucleoside analogues and expensive medical costs for many patients with Chronic hepatitis B, it is clear that the application interferon and nucleoside analogues against hepatitis b virus can’t solve the all problems in patients with CHB. The regulation Immune function to Traditional Chinese medicine therapy is undoubtedly positive prevention and treatment of Chronic hepatitis B of the icing on the cake. In recently years, there have been many Chinese medicine monomer in the clinical treatment of chronic hepatitis B, but its mechanism of regulating immune function is not very clear. HZYGJWF has come from the tutor professor Shengguoguang in under the guidance of TCM theory, clinical verification and experimental research for many years. HZYGJWF is on the basis of the party with the prince ginseng, medlar. HZYGJWF under bead, spreading hedyot is herb, poria cocos, seaweed, white mustard seed, rhizoma zedoariae, medlar, radix pseudostellariae experience side composed of eight herbs. With detoxification, phlegm, blood stasis, tonify deficiency effect. Early studies suggest that HZYGJWF may in some way, link and targets to achieve two-way regulating immune function, its mechanism of action is worth further study. This topic is the immune liver injury animal models in mice as experimental object, the study of HZYGJWF about mice of immune state, spleen cell cytokines gene mRNA expression, helper T lymphocytes Th1/Th2, toll-like receptor (TLR) and its signal transduction pathways, the influence of using computer and statistical software for data processing and comprehensive analysis, to explore the immunoregulatory effects of HZYGJWF and its mechanism. traditional Chinese medicine has important academic value to improving the level of Chronic hepatitis B of viral disease control and prevention has important scientific significance.Methods:the experiment is divided into five partsThe first part:HZYGJWF and its expanding party to Con A induction of protective immunity liver injury of mice.Clean level100kun ming mice were randomly divided into ten group. normal group (1), model group (2), prednisone group (3), HZYGF group (4), HZYGF and radix pseudostellariae medlar group (5), HZYGF and radix pseudostellariae fairy spleen group (6), HZYGF and medlar fairy spleen group (7), leaf under bead spreading hedyotis herb group (8), tuckahoe white mustard group (9) and rhizoma zedoariae group(10), each group of10, only by Con A tail vein injections made immune hepatic injury animal model.12days before the date of the mold, model group and blank group was given distilled water to fill the stomach, the rest of the group were given daily spilt, HZYGF, HZYGF add radix pseudostellariae medlar, HZYGF add radix pseudostellariae fairy spleen, HZYGF add medlar fairy spleen, hepatic lobe under bead spreading hedyotis herb, kelp, tuckahoe white mustard and rhizoma zedoariae lavage. Time to fill stomach1h after the treatment, at the end of the blank group was given saline10ml/kg tail intravenous injection, the rest of the group according to20mg/kg tail vein injection of Con A building, fasting, free drinking water. Make mould for cervical dislocation executed after8h animal liver, spleen, liver and spleen index is calculated. Specimens of liver tissue homogenate after testing ALT, AST, SOD and MDA. In addition in liver tissue paraffin embedding sectioning will been done to HE staining.The second part:the influence of HZYGJWF to Con A-induced immunological liver injury in mice serum IL-4, IL-6, INF-γ, TNF-α.Clean level40kun ming mice were randomly divided into normal group (1), model group(2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury model.12days before the date of the mold, model group and blank group was given distilled water to fill the stomach, the rest of the group were given daily over the loose, HZYGF lavage. Time to fill stomach1h after the treatment, at the end of the blank group was given saline10ml/kg tail intravenous injection, the rest of the group according to20mg/kg tail vein injection of Con A building, fasting, free drinking water. Building after8h pick eyeball blood, centrifugal file.usd10.00serum. Using ELISA method for determination of mice serum IL-4, IL-6, IFN-γ, TNF-α value.The third part:HZYGJWF to Con A-induced immunological liver injury in mice liver tissue pathological changes and the expression of NF-KP65level.Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury model. Building to put to death in mice after8h, take make ultrastructural slices of liver, the liver tissue by tem observation group the pathological changes of the ultrastructure of liver cell, at the same time take liver tissue in mice liver tissue was determined by Western blot method-the expression of NF-KP65level.The fourth part:HZYGJWF to Con A-induced immunological liver injury in mice spleen in mice group, IL-6and TNF-α mRNA.Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury in mice model. The mice spleen tissue was evaluated by RT-PCR method, IL-6, TNF-a mRNA expression.The fifth part:HZYGF sides to con A induced immunological liver injury mice liver tissue TLR4, and CD14mRNA in mice.Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injection made immune liver injury model. The liver tissue of mice was evaluated by RT-PCR method the expression of TLR4and CD14mRNA.Results:The first part:hai zhu yi liver and its expanding party to Con A induction of protective immunity liver injury of mice. Success immune liver injury in mice model was established.1Compared with normal group, model group mice liver and spleen index, liver tissue homogenate supernatant after ALT, AST andMDA content has significantly increased (P<0.05), liver tissue homogenate supernatant after the SOD level is significantly reduced. Model group liver tissue in a large number of lymphocyte infiltration, part of the portal area lymphocyte infiltration, erythrocyte sedimentation within liver sinus, a large number of liver cell nucleues is broken, scattered apoptotic body, liver steatosis in cell plasma. Liver cells around vascular cell pack loose, color becomes shallow, all loose cytoplasm of liver cells, staining obviously becomes shallow, hepatic sinus gap widened significantly, liver disappear, sometimes visible point necrosis, lymphocyte, neutrophil infiltration.2Compared with model group, control group and haizhu yi liver flavored group can decrease the mice liver and spleen index, lower ALT, AST and MDA content in liver tissue, improve the level of liver tissue SOD (P<0.05), among them with HZYGF with radix pseudostellariae, medlar more obvious.Other party HZYGF flavored group obviously reduce degree of liver pathological changes than model group.The second part:the influence of HZYGJWF to Con A induced immunological liver injury in mice serum IL-4, IL-6, INF-γ and TNF- a.1Compared with normal group, the level of serum IFN-y and TNF-a of model group mice has increased significantly with significant difference (P<0.05). the level of serum IL-4and IL-6Model group mice has decreased with significant difference (P<0.05).2Compared with model group, the level of serum IFN-γ and TNF a of control group and HZYGJWF group has lower significantly, there is significant difference (P<0.05). the level of mice serum IL-6in Control group and HZYGJWF has increased significantly with significant difference (P<0.05). the level of mice serum IL-4Control group of mice and HZYGJWF has increased a lot, there is significant difference (P<0.05).The third part:HZYGJWF to Con A-induced immunological liver injury in mice liver tissue pathological changes and the expression level of NF-KP65.1set of normal liver cells for polygon, neatly, nuclear assumes the circular or ovoid, nuclear membrane smooth level off, the chromatin evenly distributed within the box. Model group liver cell swelling, cytoplasm osteoporosis, appear incisure, two-tone cytoplasm common in myeloid body and vacuoles degeneration area, the capillary bile duct expansion, some microvilli medullary degeneration, mitochondria swelling and cristae vague, mild expansion of endoplasmic reticulum. Compared with model group, control group and HZYGJWF group liver cell swelling significantly reduce, did not see clearly the capillary bile duct expansion, mild swelling of mitochondria crest, no obvious expansion of endoplasmic reticulum.2Compared with normal group, the NF-KP65level of model group was increased significantly, statistically significant difference(P<0.05); Compared with model group, the NF-KP65level of control group and HZYGJWF group has obvious changes, statistically significant difference(P<0.05). HZYGJWF party on immunological liver injury in mice liver tissue ultra structure has obvious protective effect and reduced immune liver injury in mice liver tissue of NF-KP65level, so as HZYGJWF can protect the immunological liver injury of con A-induced.The fourth part:HZYGJWF to A sword bean protein induced immunological liver injury in mice spleen tissues in mice, IL-6, TNF-a mRNA.1Compared with normal group, model group, the level of IL-6mRNA has significantly reduced with statistically significant difference(P<0.05); Compared with normal group, the level of TNF-a mRNA of model group has significantly increased, statistically significant differences (P<0.05).2Compared with model group, the level of IL-6mRNA of control group and HZYGJWF group has increased significantly, statistically significant differences (P<0.05). Compared with model group, the level of TNF-a mRNA of control group and HZYGJWF group has dropped significantly, statistically significant difference (P<0.05).The fifth part:HZYGJWF to Con A-induced mice immune liver injury in mice liver tissue TLR4, CD14mRNA.1Compared with normal group, model group of TLR4mRNA expression level increased obviously, statistically significant difference (P<0.05); Compared with model group, control group and HZYGJWF group TLR4mRNA expression level decreased obviously, statistically significant difference (P<0.05).2Compared with normal group, model group of CD14mRNA level no obvious changes, statistics has no obvious difference (P>0.05)。 Compared with model group, control group and HZYGJWF group CD14mRNA level no obvious changes, statistics has no obvious difference.Conclusion:HZYGJWF has obvious protective effect to immune liver injury in mice by Con A induced. the mechanism may inhibit hepatoly cellular lipid per oxidation; inhibit cell inflammatory factor the release of TNF-α and IFN-γ, improve cell suppression of inflammatory factor, IL-6and IL-4release, so as it can adjust the balance between Th1/Th2; improve the activity of TLR4and Inhibiting the activity NF-KP65, reduce the happening of natural immune. |
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