| Objective:To study effect and protective mechanisms of Shenmai Injection against articular cartilage degeneration of the knee OA models in rabbit by intraarticular injection;And the clinical effect of Shen Mai injection was evaluated by WOMAC scoring standard.Methods:Forty health adult New Zealand rabbits for experiments were randly divided into four groups (normal group, model group, HA group, Shenmai group). Except the normal group, the other three groups was induced by improved Hulth’S method. Except the normal group, the other three groups were injeeted the corresponding drug by intraarticular. After 5weeks, rabbits were sacrificed by air embolism.(1)Cut apply adequate amount of articular cartilage and made ordinary optical microscope slides and transmission electron microscope slides,to observe ultrastructural articular cartilage pathology. According to the Mankin articular cartilage of trypan-blue and histological scoring system for the pathological changes of the cartilage tissue and compare half grading the differences between groups.(2) We extracted joint fluid, measured, the levels on NO and IL-1 β and observed synovial inflammation in each group.(3)We observed the mRNA expressions of MMP-3,MMP-13 and TIMP-1 in knee cartilage of OA model rabbit by RT-PCR;(4) We cut the right amount of articular cartilage of rabbit femoral condyle, and respectively measured the level of GAG and positive stain area of Collagen Type II in each group By IHC, and compared the differences between groups. Clinical research:Select patients with knee osteoarthritis from January to December 2015 in orthopaedic of our hospital, including outpatient and hospitalization for the study. Meet the doctors diagnosed with unilateral KOA and traditional Chinese medicine diagnosed as knee rheumatism is rheumatism by lacked of yang 90 cases. Using random numbers method 90 patients were randomly divided into 2 groups, each group of 45 patients.The control group was into the Shenmai group and HA group. WOMAC score evaluation before and after treatment in the treatment efficacy. Normal distribution of metering data indicate by mean+standard deviation (x±s), using paired t test to analysis the difference of patients with arthritis of the knee which using Shenmai and control group in symptoms after treatment improved, P<0.05 believes that differences between two sets of data are statistically significant. Non-normal distribution of the measurement data indicate by the median and quartile, using the Wilcoxon statistics. K-W test for determine the differences between groups, P<0.05 as a statistically significant. To compear with differences in treatment between groups.ResuIts:(1) In macroscopic view, The normal group of rabbit articular cartilage surface smooth, flexible, light blue, translucent, no crack, defect and softening. In the model group, most of the articular cartilage surface were yellow, patchy or extensive rough, erosion, and even crack, defect and softening, loss of elasticity, articular cartilage of the HA groups^ Shenmai Injection group were the basic same in pathological changes:surface was mostly yellow, patchy or diffuse slightly rough, hadn’t the obvious crack or defect;(2) The levels on NO and IL-1β of Shenmai Injection group was lower than others, and showed significant difference (P<0.05). Synovium of the HA group and Shenmai Injection group-no or mild redness, swelling, hyperplasia, and the synovitis significantly reduced compared with model group.(3)In the normal group, the mRNA expression rates of MMP-3, MMP-13 were relatively low, and TIMP-1 was relatively high;while in the model group, the mRNA expression rates of MMP-3,MMP-13 significantly increased and TIMP-1 was significantly low;After treatment, the mRNA expression rates significantly decreased in the two treatment groups. Between Shenmai Injection group and the HA group, showed significant difference after treatment(P<0.05).(4)GAG content results in each group:①The GAG content of cartilage matrix was no significant difference between the model group and the HA group(p>0.05): ②Shenmai Injection group and the HA group, model group were compared, the GAG content of cartilage matrix of Shenmai Injection group was significantly different (p<0.01); Results of positive stain area of Collagen Type II in each group: ①The positive stain area of Collagen Type II was no significant difference between the model group and the HA group(p>0.05):②Shenmai Injection group and the HA group, model group were compared, The Positive stain area of Collagen Type II of Shenmai Injection group was significantly different(p<0.01).(5) Before and after comparing the treatment of pain:After a course of treatment, the patients feel better about the knee pain in two groups, the Shenmai group and the control group Compared with before treatment, a significant difference (KW test, P= 0.001);Shenmai group before and after treatment compared with the control group score, the difference was significant (non-normal data state, using the Wilcoxon test, P= 0.000).(6)Before and after stiff treatment comparison:After a course of treatment, Shenmai group and control patients with knee stiffness score improved improved. Compared with before treatment, a significant difference (KW test, P= 0.000) in the group differences; Shen mai group before and after treatment compared with the control group score, the difference was significant (P= 0.003)(7)Daily activity before and after Treatment:After a course of treatment, Shenmai group and knee patients in the control group were improved daily activity score and the degree of improvement. Compared with before treatment, no statistically significant difference (KW test, P= 0.874) associated with the two groups; the Shenmai group scores before and after treatment with the control group, the difference was not statistically significant (data normality, paired T-test, P= 0.601).(8)Before and after treatment WOMAC total score comparison:After a course of treatment, Shenmai group and control group patients’daily activities of the knee score were improved and improved. Compared with before treatment, the difference was not statistically significant (one-way ANOVA, P= 0.155); Shenmai group and the control group before and after scores with self-heating, the difference was significant (data is being normal distribution, paired T-test P= 0.001).Conelusions:(1) Shenmai Injection could significantly reduced the levels on NO and IL-1 β in the articular cavity of the knee OA model in rabbit, and could inhibit synovitis disease.(2) Shenmai Injection could inhibit the mRNA expressions of MMP-3, MMP-13 in Knee Cartilage of OA Model Rabbit by Intraarticular Injection, and increase the mRNA expressions of TIMP-1, and delay the degeneration of articular cartilage.(3) Shenmai Injection could not only improve the GAG content of cartilage matrix,and promote the synthesis of collagen type Ⅱ.(4) Shenmai Injection could promote extracellular matrix synthesis,and degradation of newly synthesized matrix was significantly inhibited.(5) this study confirmed that Shenmai Injection in the treatment of knee osteoarthritis in the relief of pain, improve the effect of the stiffness of the joint and better than the HA.Results showed that:Under the common function of various factors in the above, Shenmai Injection could promote the synthesis of collagen type Ⅱ.increase GAG content, inhibit degradation of ECM, reduce or control inflammation of KOA, thus maintain and keep the articular cartilage normal structure and function, prevent the process of degenerative diseases of rabbit KOA model. Shenmai Injection can relieve pain and improve joint function in clinical treatment. |
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