| The pathogenesy of ankylosing spondylitis (AS) is indefinite up to now, and western medicine has no specific treatment. Although there are many new therapies, such as TNF-α blocking agent, thalidomide, pamidronate, et al, the treatment of AS still has no essential improvement. Traditional Chinese medicine(TCM) have advantages on better curative effect and less adverse effect in t AS treatment, so it is very important to study on the TCM regularity of diagnosis and treatment. In this study, we assessed the curative effect and the safe of Bushen Qiangji Granula by a randomized, controlled and single-blind clinical trial in order to acquire the strong clinical evidence to verify the therapy of invigorating the kidney and promoting the blood effective. Moreover, we design an experiment to uncover a new anti-ossification function of the therapy of invigorating the kidney and promoting the blood by using the technique of cell molecular biology.Theory study1. Reviewed and sum up the ancient TCM literature relating to AS, including the name, the etiological factor, the pathogenesis, the therapy and the prescript -tions.2. Summarized the academic experience of Professor Feng Xinghua in AS treatment. It was extracted to: the origin etiological of AS was renal deficiency and DU meridian vacuity, and the main therapy was to invigorate the kidney and strengthen DU meridian; the appearance of AS was humid heat arthralgia spasm, and the therapy was to clean the heat and damp elimination; blood stasis was also important, so we should promote blood flow during all the courses.3. Analyzed the kidney playing an important role in AS through TCM theory and modern genetics and immunology. We drew an academic viewpoint: To take, express and regulate the genetic information and reconstruct the immunological homeostasis should be closely related to invigorate the kidney in TCM.4. Emphasized the important role of blood stasis in AS process, all of exogenous pathogenic factor, kidney-essence deficiency, entrails internal damage and protracted disease may result in blood stasis. The blood stasis lay in spinalcolumn, waist, buttocks, so it is important to promote blood flow, dissolve stagnation and dredge DU meridian.Clinical studyEvaluate the effect and safe of Bushen Qiangji Granula in treating AS patients with the syndrome of renal deficiency and blood stasis by a randomized, controlled and single-blind clinical trial. The patients were treated by Bushen Qiangji Granula for six months. Applying international AS evaluation factor and TCM syndrome assessment criterion (Chinese materia medica new drug clinical research guidance), we evaluate the effect of Bushen Qiangji Granula in AS treatment. Results:? The granule alone or salicylazosulfapyridine(SASP) affiliated both had good curative effect, the total effective rate were 81.82% and 86.21%, and the total effective rate was 96.7% and 93.1% on improving the syndrome of renal deficiency and blood stasis.(2) After 6 months' treatment, Bath AS disease activity index, Bath AS function index, pain, and early morning stiffness time in patients had conspicuous improvement. Schober test, the activity of the thoracic cage, finger-ground test were improved. And ESR and CRP were improved.(3) The effect of Bushen Qiangji Granule had obvious chronergy, the effect was gradually outstanding followed by the extend of treating time.? The effect of Bushen Qiangji Granule wasn't significantly increased associated with SASP, only the work time was earlier than it alone, but the association increase the risk of adverse effect and treatment cost.(5) The effect, the safe and the compliance of Bushen Qiangji Granule was satisfactory. Also, it was taken conveniently. It's necessary to apply and exploit Bushen Qiangji Granule.In this study, we combined the ASAS factor with the TCM syndrome assessment, and synthetically assess the effect of Chinese medicine on AS disease and TCM syndrome. This study applied for the clinic the strong evidences to verify the therapy of invigorating the kidney and promoting the blood; and promote the clinical study's normalization and standardization in TCM study.Experimental studyThis part study is financed by National Natural Science Fund. Integrating TCM theory and advanced technology in modern life science, using the cultured fibroblast from AS patients capsula articularis coxae and the Chinese materia medica serum pharmacology, operating modern cell molecular biology technology, such as cell culture, flow cytometry, real-time quantitative PCR, westernblot, immunohistochemistry and so on, we uncover a new anti- ossification function of Bushen Qiangji Granule and Icariin,1. Establishment of a Finite AS Fibroblast LineUtilizing the capsula articularis coxae tissue from AS patients, we establish a biological character stationary fibroblast line. As the immunohistochemistry result was vimentin positive and ceratin negative, we identified its mesoblast origin. It was a good model in vitro to study AS ossification pathogenesy and anti-ossification drug.2. Study on AS Fibroblast Proliferation, Apoptosis and the Differentia -tion to Ossification TypeControlled with the fibroblast coming from the capsula articularis coxae of normal human, AS fibroblast grew faster from the second day after innoculated, and on the sixth day, its growing rate was faster 63.2% than normal fibroblast. Also, AS fibroblast proliferation cycle was disordered. The cell ratio of S period was obviously increased coursed by much more DNA duplicated( P<0.01), and the ratio of Go/ Gi period was decreased. PI/ Hoechst 33342 fluor dyeing showed that little apoptosis cell was found under fluor microscape, and the earlier and later apoptosis rate was lower than normal fibroblast. The result showed that the excessive proliferation, the abnormal cell cycle and restrained apoptosis maybe the important factor to accelerate AS ossification. ?Alkaline phosphatase(ALP) activity of AS fibroblast, collagen synthesis and osteocalcin(OC) secretory volume in clear supernatant liquid was obviously higher than normal fibroblast (P<0.05, P<0.01); real-time quantitative PCR showed the ossification marker gene cbfal and OC mRNA expressed obviously higher than normal fibroblast (PO.01, P<0.001), and western blot showed that cbfal protein positive expressed in AS fibroblast and negative in normal cells. The results showed that the fibroblast expressed ossification phenotype because of the ossification marker gene "reopen" , and it lead to local ossification in AS patients.3. Effect of Bushen Qianji Granule on AS Fibroblast Proliferation, Apoptosis and the Differentiation to Ossification TypeThe serum with little and middle dosage of Bushen Qianji Granule repressed AS fibroblast proliferation(P<0.05), and regulated cell cycle by holding back the cells entering S period and making the cell division arrest at Go/ Gt period. Besides, the serum with every dosage could increase the earlier and later apoptosis ratio (P<0.01, P<0.001) .The serum containing SASP had no effect on cell proliferation, cell cycle and apoptosis. Results showed that Bushen Qianji Granule had the effect on repressing AS fibroblast proliferation, regulating the cell cycle and inducing apoptosis, and it maybe one of the important mechanism of blocking up AS ossification proceeding.Then, we found the serum containing Bushen Qianji Granule could repress AS fibroblast ossification phenotypic expression. High dosage suppresed cell ALP activity, collagen synthesis and OC secretion, middle dosage suppressed cell ALP activity and OC secretion, and small dose has no repress effect on ossification phenotypic expression. The serum with high dosage suppressed AS fibroblast cbfal mRNA expression, yet middle and large dosage could obviously repress OC mRNA expression. Moreover, the serum with middle and high dosage could repress cbfal protein expression. The results told that Bushen Qianji Granule could block up the proceeding ossification of AS fibroblast through repressing cell ALP activity, collagen synthesis and OC secretion; and it was the molecular mechanism to repress AS fibroblast ossification marker gene expression.4. Effect of Icariin on AS Fibroblast Proliferation, Apoptosis and the Differentiation to Ossification TypeEpimedium herb was the essential component in Bushen Qiangji Granule, and Icariin was the main component of Epimedium herb. Icariin had no obvious effect on AS fibroblast proliferation, cell cycle and apoptosis. But, icariine could repress AS fibroblast ossification phenotypic expression. Little dosage depressed collagen synthesis, middle depressed ALP activity and OC secretion, and high depressed ALP activity, collagen synthesis and OC secretion. Also, icariin with every dosage depressed the expression cbfal mRNA and the protein, and the effect was stronger followed by the increased drug density. Also, small and large dose could repress the expression of OC mRNA (P< 0.01) . The results showed that icariin had the effect on AS fibroblast ossification phenotypic expression, and its... |
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