| Objective:To study Huangqiguizhiwuwu decoction’s effect in treating osteoarthritis deficient in yang & excessive in yin, based on the validity of the rats model, in order to extend Huangqiguizhiwuwu decoction’s applying field, and then do further study about Huangqiguizhiwuwu decoction’s mechanism in treating osteoarthritis’(OA).Method:50 rats, divided into 5 groups, which are control group, model group, Huangqiguizhiwuwu decoction group of 7 days, and Huangqiguizhiwuwu decoction group of 14 days, celecoxib group at random. The specific methods rebuilding osteoarthritis model deficient in yang & excessive in yin as follows:Except the control group, other 4 groups are made for OA model which is deficient in yang and excessive in yin by joint plaster fixation and physical stimulation methods (feed every two days 6 weeks and swim 2 weeks). Then, Huangqiguizhiwuwu decoction group of 7 days, Huangqiguizhiwuwu decoction group of 14 days and celecoxib group are gastric respectively according to their weight 6 mL · Kg-1, and Huangqiguizhiwuwu decoction group of 7 days is drugged 5 g·mL-1 for 7 days from the first day of the 6th week in rebuild osteoarthritis model, Huangqiguizhiwuwu decoction group of 14 days drugged 5 g·mL-1 for 14 days from the first day of the 5th week in rebuild osteoarthritis model, while celecoxib group is drugged 3.47 g·L-17 days from the first day of the 5th week in rebuild osteoarthritis model. The normal group and OA model group are gastric by distilled water equally from the first day of the 5th week in rebuild osteoarthritis model.6 weeks later, collect rats’serum, plasma and right knee joint. Observe every group’s general condition, calculate the weight and its swollen of the knee joint. Detect CD4+, CD8+ in the serum by FCM, and test the content of SOD, IL-1β, TNF-a, IL-4, PGE2, HIF-1α, IGF in serum and TXB-2,6-keto-PGF1α by ELISA. Then using microscope to observe the pathological change in knee joint and iNOS, TGF-1β in cartilage and synovium, and uPA, PAI-1 in vein of subchondral bone,and collagen of type 2 in Cartilage.Results:(一) Huangqiguizhiwuwu decoction’s treating effect in OA(1)Compared with the control group, model group is in bad mental state, and its skin and hair lost lustre. Rats eat less and has loose stool. The tail has bruise. The limber is swollen, even ulcerate. They can’t move flexibly, or appear claudication. Compared with model group, Huangqiguizhiwuwu decoction group of 14 days and celecoxib group have smooth fur, and no bruise appear in tail. The limber can move better than model group, and have no claudication. Huangqiguizhiwuwu decoction group of 7 days move little, fur has no gloss but better than the model group. The tail has slight bruise. Limber is less swollen than model group, move inflexibly, and still has claudication.(2)The weight is decreased in OA model group compared with control group, and weight in Huangqiguizhiwuwu decoction 14 days group increased compared with OA model group, while Huangqiguizhiwuwu decoction 7 days group and celecoxib group have no significant difference compared with OA model group. The swollen degree of lower limber in OA model group increased compared with the control group, and other 3 gastric group decreased compared with the OA model group,3 gastric groups have no significant difference.(3)Collagen of type â…¡ in OA model group decreased compared with control group, and the collagen of type â…¡ in other 3 gastric group increased compared with model group. Among the 3 gastric group, celecoxib group’s type â…¡ Collagen increased than Huangqiguzihiwuwu decoction 7 and 14 days groups, and there is no difference between Huangqiguizhiwuwu decoction for 7 days and 14 days.(二) Huangqiguizhiwuwu decoction’s influence in treating OA about immune response.(1)The percentage of CD4+ and CD8+ are lower compared with control group, and the percentage of CD4+ and the ratio of CD4+/CD8+ in huangqiguizhiwuwu decoction 14 days group and celecoxib group increased compared with OA model group, while CD8+ have no significant difference among these 5 groups. In the 3 gastric group, the percentage of CD4+ and the ratio of CD4+/CD8+ have no significant difference.(2)The content of IL-1β, TNF-a in OA model group decreased than control group, and increased in 3 gastric groups than model group. meanwhile, there is no significant difference in 3 gastric groups. The content of PGE2, HIF-1α, iNOS in OA model group are higher than control group. Among the 3 gastric groups, the content of PGE2 and iNOS are lower, and HIF-1α in Huangqiguizhiwuwu decoction 14 days group is lower than OA model group,the other 2 groups have no significant difference. In the 3 gastric groups, the level of iNOS huangqiguizhiwuwu decocotion 7 days group is highest than huangqiguizhiwuwu decocotion 14 days group and celecoxib group, and PGE2 has no significant difference, and HIF-1α has no difference in 5 groups.(3)The content of IL-4 increased than control group, TGF-1β in OA model group decreased than control group, and the content of IL-4 decreased in 3 gastric groups compared with the model group and TGF-1β increased than model group. In the 3 gastric groups, IL-4 and TGF-1β have no significant difference. IGF has no significantly difference among 5 groups.(三)Huangqiguizhiwuwu decoction’s influence in treating OA on blood stasis.(1)TXB-2 of OA model group in plasma is higher than control group,6-keto-PGFla of model group is lower than control group, and the ratio of TXB-2/6-keto-PGFla increases in model group compared with the control group. TXB-2 and the ratio of TXB-2/6-keto-PGF1α in 3 gastric groups are lower than OA model group,6-keto-PGF1α in 3 groups are higher than OA model group. Among 3 gastric groups, TXB-2, 6-keto-PGFla and its ratio have no significant difference.(2)The expression of uPA and PAI-1 at subchondral bone’s vessel in OA model group are higher than control group, and 3 gastric groups are lower than OA model group. Among the 3 gastric group, expression of PAI-1 of Huangqiguizhi wuwu decoction 14 days group is lowest compared with Huangqiguizhiwuwu decoction 7 days group and celecoxib group.(å››)Huangqiguizhiwuwu decoction’s influence in treating OA about SOD.The lever of SOD in OA model is lower than the control group. SOD in 3 gastric groups increased compared with OA model group. In 3 gastric groups, the lever of SOD in Huangqiguizhiwuwu decoction 14 days group is significantly higher than celecoxib group, but huangqiguizhiwuwu decoction 7 days group has no significant difference with celecoxib group.Conclusion:(1)Huangqiguizhiwuwu decoction decreases the weight and swollen of OA model, and the cartilage turns into smooth and the cell of cartilage is arranged in order, meanwhile, it reduce pannus in subchondral bone vessle. We noticed that huangqiguizhiwuwu decocotion 7 days group has little influence to the weight, swollen than huangqiguizhiwuwu deocoction 14 days group, but the condition of enlargeable collagen fiber, pannus and inflammatory cells is not as serious as Huangqiguizhiwuwu decoction 14 days group, this indicates that huangqiguizhiwuwu decocotion has treating function for OA which is deficient in yang & excessive in yin, and its function correlative with the treating course.(2)After gastric, the percentage of CD4+and the ratio of CD4+/CD8+decreased, it means Huangqiguizhiwuwu decoction has effects on OA by immune response. Meanwhile, the level of IL-1β and TNF-a in OA model group increased, and the level of IL-4 decreased, it may attributed to the immune suppression, so that it stimulates stress response. Huangqiguizhiwuwu decocotion decreases the lever of PGE2, iNOS, and HIF-1α, and increases the lever of TGF-1β, this reflects OA has influence to cytokines including PGE2, HIF-la, and iNOS which act as decompositive cytokine, and TGF-1β which acts as compositive cytokine, and Hauangqiguizhiwuwu decoction 14 days group’s tendency is better than 7 days group. In other words, it means by changing of these factors’level, the lesion of cartilage and proteogly can be suppressed, so as to the apoptosis of cartilage. In a word, huangqiguizhiwuwu decocotion may treat OA by immune response and it has positive correlation with time.(3)Huangqiguizhiwuwu decoction 7 days and 14 days groups both can decrease the level of TXB-2, and the ratio of TXB-2/6-keto-PGFla. The expression of u PAå'Œ PAI-1 in subchondral bone vessle are also decreased in these two groups. In the 4 indexes, PAI-1’s level in Huangqiguizhwuwu decoction 14 days group is lower than 7 days group, and the other 3 indexes have no difference between the 2 groups, and 14 days group’s tendency in effecting the 3 indexes is better than 7 days group. It means long-time using of Huangqiguizhiwuwu decoction protect cartilage cell and can treat OA by means of relieving the hypercoagulable state, so that the local blood circulate is improved, and it further interacts with cytokine IL-1β, TNF-α, HIF-1α, etc, so they may reduce joint swelling, and pressure in the bone to inhibit the lesion of collagen, and restructure of subchondral bone.(4)The fact that Huangqiguizhiwuwu decoction can increase the level of SOD in serum,a and Huangqiguizhiwuwu decocotion 14 days group has better tendency than 7 days group. It means huangqiguizhiwuwu decoction’s function is relative with oxygen radical, and it correlated with irradiation time. |
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