Study On The Intervention Effect And It’s Mechanism Of Huayujiedufa On Thrombin Toxicity Mediated Inflammatory Responses In MCAO Rats

Objective:To observe the influence of Huayujiedufa on the expression of thrombin and its receptor (PAR-1) and monocyte chemoattractant protein (MCP-1) and nuclear factor κ B (NF-κ B) of brain tissue in MCAO rats, and the expression of macrophages (BTM) and other inflammatory cell infiltration. Explore the essence of AIS "Ecchymosis" pathogenesis and the intervention of Huayujiedufa based on the toxicity of thrombin.Methods:The84healthy adult male SD rats were selected. The rats were randomly divided into7groups:sham-operated group (SG),Model group(MG),Huoxuehuayufa group (HG),Qingrejiedufa group (QG),Huayu-jiedufa group (HYJDF),PDTC group (PG),Argatroban group (AG),12rats in each group. The model was reproduced by occluded middle cerebral artery, and the corresponding drugs were given at lhour before operation and12hours after operation. Samples were taken for medical analysis24hours later after operation. Fluorescent quantitative real-time PCR and Western blot were used to detect mRNA and protein expression levels of Tb, PAR-1, MCP-1and NF-κ B, and detecting ischemic brain tissue inflammatory cell infiltration using HE method.Results:1. The rats in the sham operation group had no obvious symptoms of nerve function defect,but the model group showed obvious neurological defect symptoms(P<0.05). Compared with the model group, neural function defect symptoms of the treatment group were light(P<0.05); The neurological deficit scores between groups were statistically significant (P<0.05), the treatment groups with different therapeutic effects, which Argatroban team the best, followed by Huayujiedufa group, Huoxuehuayufa group worst.2. The neuron was integrity, nucleus was round, nucleolus was clear, chromatin distribution was uniform in brain tissue of sham operation group, and no obvious inflammatory cell infiltration. The brain tissue of rats in model group showed severe ischemic changes, necrosis of neurons were broken or disappeared, the cell swelling, deformation, nuclear were pyknosised and deep stained, nucleolus wasn’t obvious, chromatin was reticulate, nuclear membrane thicking, cells arranged in disorder, inflammatory cell infiltration. The treatment group rats was mild ischemic change, cells with mild edema, few nuclear were pyknosised and deep stained, nucleolus wasn’t obvious, edema stromal cells, cells arranged unclear, and a small amount of inflammatory cells infiltration. The number of inflammatory cells in Huayujiedufa group and Argatroban group were the least.3. Compared with the sham operation group,the expression of thrombin protein in brain tissue of rats in model group and treatment group was significantly increased, the difference was statistically significant (P<0.05). Compared with model group,its expression was decreased in each treatment group(P<0.05). The comparison between treatment group, Argatroban was the least (P<0.05),Huoxuehuayufa group and PDTC group than the other groups were higher (P<0.05), the difference between the two had no statistical significance (P>0.05). There no statistical significance between Huayujiedufa group and Qingrejiedufa group (P>0.05).4.The single effect of Huoxuehuayufa or Qingrejiedufa on the thrombin protein expression were statistically significant (P<0.05),so did Huayujiedufa group which based on the two method, and had their interaction of the two method.5. Compared with the sham operation group,the expression of PAR-1mRNA in brain tissue of rats in model group and treatment group was significantly increased, the difference was statistically significant (P<0.05). Compared with model groups,its expression was decreased in each treatment group (P<0.05). The comparison between treatment groups, Huoxuehuayufa group and PDTC group than the other groups were higher (P<0.05), the difference between the two had no statistical significance (P>0.05). There no statistical significance between the other groups(P>0.05). The comparison results between the groups in the expression of PAR-1protein were same to the expression of PAR-1mRNA.6. Compared with the sham operation group,the expression of MCP-1mRNA in brain tissue of rats in model group and treatment group was significantly increased, the difference was statistically significant (P<0.05). Compared with model group,its expression was decreased in each treatment group (P<0.05). The comparison between treatment groups, Argatroban group was the lowest (P<0.05), the expression of Huayujiedufa group and Qingrejiedufa group gradually increased, the difference between the two had statistical significance (P<0.05). Huayujiedufa group and PDTC group were higher than the other groups (P<0.05), the difference between the two had no statistical significance (P>0.05).The expression results of MCP-1protein:comparison between groups and sham operation group and the model results were the same to gene’s expression. The comparison between treatment groups, Argatroban group and Huayujiedufa group was the lowest (P<0.05),the difference between the two had no statistical significance (P>0.05). Qingrejiedufa group and PDTC group and Huoxuehuayufa group gradually increased, the difference between the three had statistical significance (P<0.05).7. Compared with the sham operation group,the expression of NF-κ B mRNA in brain tissue of rats in model group and treatment group was significantly increased, the difference was statistically significant (P<0.05). Compared with model group,its expression was decreased in each treatment group (P<0.05). The comparison between treatment groups, PDTC group was lowest (P<0.05), Huayujiedufa group and Qingrejiedufa group gradually increased, the difference between the two had statistical significance (P<0.05). Argatroban group and Huoxuehuayufa was the highest, the difference between the two had no statistical significance (P>0.05).The expression results of NF-κ B protein:comparison between groups and sham operation group and the model results were the same to gene’s expression. The comparison between treatment groups, PDTC group andHuayujiedufa group were the lowest(P<0.05), the difference between the two had no statistical significance (P>0.05). Qingrejiedufa group Argatroban group, Huoxuehuayufa group gradually increased, the difference between the three had no statistical significance (P>0.05).8. In the brain tissues of rats in all groups, the protein content of PAR-1, MCP-1, NF-kappa Bprotein content and thrombin protein content showed a positive linear correlation (P<0.05).Conclusion:Enhanced in rats brain tissue damage and expression of thrombin and obvious inflammatory reaction in acute middle cerebral artery occlusion,and the inflammatory reaction was significantly related to expression of thrombin and its receptor. The Huayujiedufa has obvious effect on thrombin and its receptor expression and inflammatory reaction, but the Qingrejiedufa and Huayujiedufa has more significant effects, and Huayujiedufa and Qingrejiedufa have obvious synergistic effect on the inhibition of thrombin and its receptor expression and inflammatory responses.