| Ankylosing Spondylitis (AS) is a chronic inflammatory disease, and its pathologic changes include inflammation, bone destruction and new bone formation. The inflammation mainly occurs around the spine and the sacroiliac joint insertion sites of tendons or ligaments. With the development of AS, Inflammation progressively damages the bone cortex, and then ectopic new bone formation gets a beginning. And after a few circles of the process, we can see the end characterized by the fusing sacroiliac joints, square centrums, and the calcific tendons and ligaments. That’s to say, disability is the end. So, inflammation is the first factor that ruins the body, and the control of inflammation must be made. IL-17is an important cytokine that combines to its receptors on the surface of mainly different cells and then medicates many different pathways of inflammation of AS. And IL-23/Th17pathway is an important pathway for the differentiation and proliferation of Thl7and generation and secretion of IL-17. After combining to its receptors on the differentiated Thl7cells, IL-23activate the JAK2/STAT3signal pathway, and then the STAT3proteins are phosphorylated. Two phosphorylated STAT3will be a homodimer and the homodimer shall be transferred into the nuclear to start the RORc related gene that for differentiation of Thl7and start the IL-17related gene that for generation of IL-17. So, IL-23/Th17pathway plays an important role in AS and it can be the therapeutic target.The active stage of AS can be seen in the early or middle stage. During this period, the inflammatory levels always are high and the clinic syndromes always are worse. And treatment will be important. Active AS always has a series of worse syndromes, mainly showing us humid heat and stagnated blood. And the etiology and pathogenesis is deficiency fo kidney, humid heat and stagnated blood. So, the treatment shall be to clear heat, eliminate dampness, and activate blood and dissolve stasis. In the our completed research, we found that Qing-Re-Huo-Xue therapy showed a good effect on treatment of AS, especially on improving joint movement degree and controlling inflammation. But we have no idea about the mechanism of treatment of AS of Qi-Re-Huo-Xue therapy. So, we establish these assays to identify that IL-23/Th17pathway is the therapeutic target when under Qing-Re-Huo-Xue therapy, and Qing-Re-Huo-Xue therapy inhibits the expression of IL-23, the activation of JAK2/STAT3signal pathway, and the expression of RORc to stop the differentiated of Th17and generation of IL-17, and then the inflammation of AS will be controlled.Experiment One The separation of PBMC and Recovery of frozen PBMCObjective:To obtain PBMC and peripheral serum for experiments by separating the PBMC form peripheral blood and recovering the frozen PBMC.Method:A total of30patients who met the1984New York criteria (BASDAI score>4) and a total of30healthy people participated in the experiments. Ficoll-hypaque density-gradient centrifugation method were used to obtain PBMC. And conventional frozen cells recovery method were used to recover the frozen PBMC.Outcome:The PBMC and peripheral serum were obtained for experiments.Conclusion:Ficoll-hypaque density-gradient centrifugation method is a general method to separate peripheral mononuclear cells and cell frozen method is important for reservation of PBMC. When prepared for experiments, PBMC would be unfrozen.Experiment Two The expression level of IL-17and IL-23in serum of AS in active stage. Objective:IL-17is an important inflammatory cytokine medicating the inflammation in AS and IL-23plays a critical role in medicating generation and expression of IL-17. We established the experiments by testing the concentration of IL-17and IL-23in AS serum and the normal one to identify the expression level of IL-17and IL-23in AS serum.Method:ELISA was used to test the concentration of IL-17and IL-23in the serum of AS In active stage.Outcome:Both of the expression levels of IL-17and IL-23in the serum of AS in active stage were significantly higher in serum of AS patients than the normal ones (IL-17:p<0.001; IL-23:p<0.001)Conclusion:IL-17is proved that it can medicate inflammation from a few pathways and it is an important cytokine in medicating AS inflammation. High expression level of IL-17in serum means that it plays a significant role in AS inflammation. IL-23is a cytokine showing a critical role in medicating the expression of IL-17, and high level of IL-23in AS serum maybe lead to significantly high level of IL-17.Experiment Three The expression level of IL-17and IL-23after taking Qing-Re-Huo-Xue therapy.Objective:Qing-Re-Huo-Xue therapy shows a good therapeutic effect in controlling AS inflammation. Experiments were established to identify the effect of Qing-Re-Huo-Xue therapy on the expression of IL-17and IL-23.Method:Elisa was used to test the concentration of IL-17and IL-23by ELISA method.Outcome:For3months’treatment of Qing-Re-Huo-Xue therapy, the levels of IL-17and IL-23were both lower than before taking this therapy (IL-17:p<0.05; IL-23:p<0.05). For6months’treatment, the levels of IL-17and IL-23were also both significantly lower (IL-17: p<0.01; IL-23:p<0.01). And for6month’s treatment, the level of IL-17was much lower than that treatment for3months (p<0.05), but no difference for IL-23(p>0.05)Conlusion:Qing-Re-Huo-Xue therapy showed a good therapeutic effect on AS, especially on controlling AS inflammation and improving the pain and function of joints. With the treatment of Qing-Re-Huo-Xue therapy for3and6months, the levels of IL-17and IL-23both got lower, indicating that IL-17and IL-23are two therapeutic targets for Qing-Re-Huo-Xue therapy and Qing-Re-Huo-Xue therapy can inhibit the expression of IL-17and IL-23. The possible mechanism is that Qing-Re-Huo-Xue therapy inhibits the expression of IL-23to stop the expression of IL-17.Experiment Four The activation degree of JAK2/STAT3signal pathway in AS patients’PBMCsObjective:JAK2/STAT3signal pathway plays an important role in differentiation of Th17and generation of IL-17. Experiments were established to identify that activation degree of JAK2/STAT3signal pathway in AS patients’PBMCs by testing the protein expression levels of IL-23R, JAK2, pJAK2, STAT3, pSTAT3and RORc, and mRNA expression level of RORc.Method:The protein expression levels of IL-23R, JAK2, pJAK2,.STAT3, pSTAT3and RORc, the mRNA expression level of RORc with Western Blotting and Realtime PCR.Outcome:Compared to the healthy ones, the protein level of IL-23R was higher in PBMC of AS in active stage(p<0.05). Though no differences were found in the protein level of JAK2and STAT3(JAK2: p>0.05; STAT3:p>0.05), the protein level of pJAK2and pSTAT3were both significantly higher in AS PBMC than the normal ones (pJAK2:p<0.001;STAT3:p<0.001),and pJAK2/JAK2and pSTAT3/STAT3got the same results (pJAK2/JAK2:p<0.001; pSTAT3/STAT3:p<0.001). The protein level and mRNA level of RORc were both significantly higher(protein level:p<0.001; mRNA level:p<0.001).Conclusion:JAK2/STAT3signal pathway is proved that it plays a critical role in medicating the differentiation of Th17and generation of IL-17. In the AS patients of active stage, Both of IL-23level and the activation degree of JAK2/STAT3were significantly high. This results leads to an outcome that the level of IL-17were extremely high. And it is the molecular mechanism of that how the JAK2/STAT3signal pathway medicates the AS inflammation.Experiment Five the activation degree of JAK2/STAT3signal pathway in AS patients PBMC with treatment of Qing-Re-Huo-Xue therapy.Objective:Qing-Re-Huo-Xue therapy shows a critical role in controlling AS inflammation, especially in inhibiting the expression level of IL-17. And JAK2/STAT3signal pathway play an important role in medicating generation and secretion of IL-17. Experiments were established to identify the activation degree of JAK2/STAT3signal pathway with the treatment of Qing-Re-Huo-Xue therapy for3and6months.Method:The protein expression levels of IL-23R, JAK2, pJAK2, STAT3, pSTAT3and RORc, the mRNA expression level of RORc with Western Blotting and Realtime PCR.Outcome:With the treatment for3months, the protein level of IL-23R was lower than the PBMC form patients in active stage(p<0.05). No differences were found in the protein levels of JAK2and STAT3(JAK2: p>0.05; STAT3:p>0.05), but the protein levels of pJAK2and pSTAT3were much lower (pJAK2:p<0.05; STAT3:p<0.05), and pJAK2/JAK2and pSTAT3/STAT3got the same results(pJAK2/JAK2:p<0.05; pSTAT3/STAT3: p<0.05) after3months treatment. The protein level and mRNA level of RORc were also lower after3months treatment (protein level:p<0.05; mRNA level:p<0.05). With the treatment for6months, the protein level of IL-23R was lower than the PBMC form patients in active stage(p <0.05). Still no differences were found in the protein levels of JAK2and STAT3(JAK2:p>0.05; STAT3:p>0.05), but the protein levels of pJAK2and pSTAT3were significantly lower (pJAK2:p<0.01; STAT3:p <0.01), and pJAK2/JAK2and pSTAT3/STAT3got the same results (pJAK2/JAK2:p<0.01; pSTAT3/STAT3:p<0.01) after6months treatment. The protein level and mRNA level of RORc were also lower after3months treatment (protein level:p<0.05; mRNA level:p<0.01). And after6months’s treatment, no difference of IL-23R protein level was found compared to3month’s treatment (p>0.05), but phosphorylated JAK.2and STAT3were both lower (pJAK2:p<0.05; pSTAT3:p<0.05), and the protein level of RORc was also lower (p<0.05) though there was no difference for the mRNA level of RORc (p>0.05)Conclusion:The activation degree of JAK2/STAT3and expression level of IL-17and IL-23were inhibited after Qing-Re-Huo-Xue therapy for3and6months, indicating that IL-23、IL-17and JAK2/STAT3signal pathway are the therapeutic target for Qing-Re-Huo-Xue therapy. The possible molecular mechanism of Qing-Re-Huo-Xue therapy used in controlling AS inflammation is to inhibiting the expression level of IL-23and the activation degree of JAK2/STAT3signal pathway to stop the expression of IL-17. |
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