| Objective:Study the pain relief mechanism of HQGZWWT（Huangqi Guizhi Wuwu decoction）on neurotransmitter and inflammatory reaction of osteoarthritis based on the study of its analgesic effect on hot plate test and torsion reaction of mice,so as to provide experimental basis for the analgesic effect of HQGZWWT on osteoarthritis.Method:Experiment 1:Female SPF level’s CD-1 mice were divided into normal group and model group according to random number table method.The model mice were prepared with exhaustive swimming in cold water and cool living environmental stimulating for 2 weeks.Then the mice in model group were randomly divided into hot plate model group and HQGZWWT group,HQGZWWT group was administrsted with 5.2 g·kg-1 for 7 days,the other two groups were treated with the same dose of distilled water,the hot plate experiment were made after the treatment,and the potential time on mouse’s licking toe was detected in 45,90,135 and 180 min after the last administration.The mice were also detected on torsion’s potential time and frequency after modeling with the same method combined with 0.6%glacial acetic acid solution.Experiment 2:Female SPF grade SD rats were randomly divided into model group and normal group.The rats in model group were prepared by cold-solid method combined with ice-water stimulation,low-temperature external stimulation,and hunger-saturated diet.After modeling,the rats of successful modeling were randomly divided into model group,HQGZWWT high,medium and low doses groups,celecoxib positive control group.All drug groups were respectively administrated with corrsponding drugs(11.7 g·kg-1,5.85 g·kg-1,2.93 g·kg-1,0.018 g·kg-1)for 28 days,once a day.Rats in normal and model groups were lavaged with the same dose of distilled water for 28 days.During the experiment,the symptoms of rats in each group were observed and the arthritis index was evaluated.The body weight,paw temperature and the width of right knee joint of rats in each group were measured regularly,and the degree of joint swelling was calculated.After treatment,serum,brain,knee bone joint and synovium were separated.After that,IL-1,IL-6,SP,5-HT and DA content in serum,and COX-2,DA,NGF,5-HT and SP content in brain were detected by ELISA method,protein and gene expression of NGF and COX-2 in synovium and brain tissue were tested by immunohistochemical,Western blotting and QPCR.HE staining was used to observe the pathological changes of knee joint and brain tissue.Results:（一）Effect of HQGZWWT on Pain in Mice （1）Study on analgesic effect of HQGZWWT on mice hot plate pain After administration,the potential licking time of mice in each group was compared.Compared with the normal group,the potential licking time of mice in the model group was reduced in each time period,while there was significant difference at 180 min（P<0.05）.There was an increase in the dosing group at each time period,and the difference was significant at 180 min（P<0.05）.Compared with the model group,the drug administration group showed an increase in each time period,and the difference was significant at 90 min,135 min and 180 min（P<0.05）.（2）Study on analgesic effect of HQGZWWT on acetic acid writhing in mice After administration,the incubation period and number of writhing in each group were compared.Compared with the normal group,the number of writhing in the treatment group was decreased（P<0.05）.Compared with the model group,the number of body twisting was reduced in the drug administration group（P<0.05）.（二）Experimental study of HQGZWWT on rats with osteoarthritis caused by Yang-deficiency and cold coagulation（1）Compared with rats in the normal group,rats in the model group which had sticky and rough were listless.The hair of both lower limbs was badly shed,and the exposed skin was dark purple.The limbs were not active,and obviously swelling,even showed ulceration.Diet in model rats was reduced,and the stool was not formed.Compared with the model group,celexib group and the rats in the medium and high-dose groups of HQGZWWT showed mental recovery,hair color restoration,occurred hair growth on both lower limbs,basically unrestricted movement,increased diet and stool formation.In the low-dose group,the spirit of HQGZWWT improved slightly,and the hair color restored luster,and the activity and swelling of both lower limbs were limited,but it was lighter than that of the model group.（2）Pathological sections of the structure of the knee joint of rats in each group showed that the surface of the knee joint in the normal group was flat without any defect,and chondrocytes were arranged neatly.A small amount of vascular tissue or vascular endothelial cells were observed in the synovium tissue.And there were defects on the surface of the articular cartilage in the model group,the chondrocyte layer was thinner than that in the normal group,and the number of cells was reduced.There were more small blood vessels in the model group,and pannus was more serious.The surface of the cartilage in the celecoxib group was blurred,the number of chondrocytes was more than that in the model group,and chondrocyte division was observed.The cartilage surface of each dose group of HQGZWWT was relatively flat,showing a large number of proliferation and division stage’s chondrocytes,and the distribution of chondrocytes was less.Meanwhile,its distribution of synovial layer cells was less than that in model group,even though the distribution of small blood vessels was still visible,and the distribution of synovial inflammatory cells was less.（1）Compared with the normal group,the arthritis index,the toe temperature and swelling degree of the model group showed an increasing trend（P<0.05）,while the body weight decreased（P<0.05）.Compared with the model group,the weight of each drug group was increased,and significantly increased in celexib and the low-dose group of HQGZWWT（P<0.05）,while the arthritis index was decreased,the toe temperature and the degree of joint swelling were decreased in the celexib group and the medium-dose group of HQGZWWT（P<0.05）.（2）Effect of HQGZWWT on inflammatory related factors in Yang-deficiency cold-coagulated osteoarthritis rats Compared with normal group,the levels of IL-6,IL-1 cox-2 in serum were increased significantly（P<0.05）,and the cox-2 protein expression in synovial membrane and rain tissue were significantly higher（P<0.01）.Compared with model group,the levels of serum IL-1,IL-6,cox-2 were decreased significantly in treatment groups（P<0.05）,the cox-2 protein expression in the synovial tissue volume significantly reduced（P<0.05）,and the positive control group,low,medium dose group of cox-2 protein expression decreased,Cox-2 protein expression was significantly reduced in the high-dose group（P<0.05）.（3）Effects of HQGZWWT on neurotransmitter factors in rats with osteoarthritis caused by cold coagulation and Yang deficiency Compared with normal group,the levels of SP,5-TH,DA content in serum of model group significantly increased（P<0.05）,the NGF protein expression in synovial membrane and brain tissue significantly higher（P<0.01）.Compared with the model group,the serum 5-TH,DA decreased significantly（P<0.05）,the level of serum SP in celecoxib and huangqi guizhi wuwu decoction group significantly lower（P<0.05）;In the brain homogenate test,compared with the normal group,the contents of DA,5-HT,SP and NGF were all increased in the model group.Compared with the model group,the contents of DA,5-HT and SP were significantly decreased in the treatment group（P<0.05）,and the index contents of NGF rats in the treatment group showed a decreasing trend（P>0.05）.In synovial tissue,compared with normal group,the NGF protein expression in model group increased significantly（P<0.01）;Compared with model group,the NGF protein expression in treatment groups significantly reduced（P<0.05）;Compared with the model group in brain tissue,the NGF protein expression in positive control group,low,and medium dose group of huangqi guizhiwuwu decoction decreased,and that in high dose group significantly reduced（P<0.05）.Conclusion:（1）HQGZWWT has a good analgesic effect on cold and dampness model mice,The analgesic effect gradually increased over time within three hours after administration,the results showed that HQGZWWT could relieve the pain in the model mice.（2）HQGZWWT significantly increased the body weight of rats,reduced arthritis index,toe temperature,swelling degree of knee joint,and resulting in the smooth joint surface,orderly alignment of chondrocytes,reduced inflammation and macrophages,and more complete subchondral bone structure.After administrating of HQGZWWT,the cartilage surface is relatively flat,the distribution of chondroinflammatory cells was less,and the number of synovium layer cells was reduced,indicating that HQGZWWT has a certain effect on the treatment of osteoarthritis pain in rats with Yang deficiency and cold coagulation.（3）IL-1,IL-6 and COX-2 content in serum were lower than model group,indicating that the analgesic effect of HQGZWWT is related to the inhibition of inflammatory cytokines,which may relieve pain by reducing the release of inflammatory factors through immunomodulation method.The COX-2 expression was significantly increased in synovial tissue,and a small amount of expression was found in brain homogenate,（4）SP,5-TH and DA content in serum and COX-2,DA,5-HT and SP content in brain were decreased after HQGZWWT administrating,indicating that the analgesic effect of HQGZWWT could be related to neurotransmitter.However,the NGF of HQGZWWT has no significant effect,even with lower expression after HQGZWWT administrating,the mechanism needed further study.In the synovium and brain tissue,compared with the normal group,the expression levels of NGF gene and protein in the model group were significantly increased（P<0.01）,and the expression levels of NGF gene and protein in the model group were significantly decreased（P<0.05）,indicating that HQGZWWT could reduce the production of nerve pain cytokines. |
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