| Objective: Tongfeng Qingxiao Formula(TQF)is a traditional Chinese medicine(TCM)with the functions of invigorating spleen and dampness,clearing heat and detoxifying,dispelling wind and relieving pain,promoting blood circulation and detumming.It has a significant effect on improving the clinical symptoms of gout and reducing the level of uric acid,but its mechanism of action is still unclear.Based on the rat model of gouty arthritis(GA)induced by dampness-heat,the TLRs signaling pathway was used as the starting point to explore the mechanism of action of TQF in the treatment of gout caused by dampness-heat,and to evaluate the therapeutic effect of TQF.To provide theoretical basis for the treatment of gout in Chinese medicine.Methods: Male SD rats were fed adaptive feeding for one week and randomly divided into 4 groups with 10 rats in each group,including normal group,damp-heat accumulation syndrome GA group(model group),TQF intervention group(Chinese medicine group)and diclofenac sodium positive control group(Western medicine group).The normal group fed ordinary diet and purified water,and the other three groups fed high-fat and high-sugar diet and honey water.The model group was given liquor and oil alternately on alternate days from day 1 to day 14,put into the artificial climate chamber with high temperature and humidity from day 8 to day 21,and injected monosodiumurate(MSU)crystal suspension into the right posterior ankle joint cavity on day 18(normal group was injected with normal saline).On the basis of model group,Chinese medicine group and Western medicine group were respectively given TQF and diclofenac sodium from 14 to 21 days.The therapeutic effect of TQF was evaluated by observing the mental state,hair,diet and water quantity,tongue image,urine and feces,weight change,gait and histopathology of rats.The expression levels of NLRP3 m RNA,TLR2/4,My D88 and NF-κB m RNA were detected by real-time quantitative PCR(q PCR).The expression levels of TLR2/4,My D88 and NF-κB protein were detected by Western blotting(WB).Results: 1.The general conditions of the rats in each group were evaluated as follows:(1)The tongue image of the rats in the normal group was normal with glossy hair;Limbs active,walking normally,spirited;Food and water intake is normal,stool is granular,urination is clarified.(2)Compared with the normal group,the tongue coating of rats in the model group was yellow and greasy,and the hair was yellow and coarse and wet;The right hind ankle joint is obviously red,swollen and febrile.When standing,the right hind limb is bent.When walking,the toe point is even off the ground,showing a three-legged gait,which is tender,lethargy,slow reaction and poor mental state.Decreased food intake and water intake,increased frequency of defecation,loose loose discharge,perianal pollution,fishy odor,red yellow urine and small volume.(3)Compared with model group,tongue quality in Chinese medicine group changed from yellow and greasy to red,and hair color did not show obvious yellowing.The joint of the right hind foot first showed swelling and then detumescence,the detumescence speed was slightly lower than that of the western medicine group,the foot was on the ground or the sole of the foot when walking,and the mental state was significantly improved;Increased food intake and water intake,reduced stool frequency,gradually formed,no odor,urine more clear;(4)Compared with the model group,the right posterior foot in the western medicine group showed swelling first and then reduced swelling rapidly.A slight improvement in mental state;Increased number of stools,loose stool,individual blood stool.2.Results of body weight increase of rats:(1)The body weight of rats in the normal group showed a curve increase.The body weight of rats in model group,Chinese medicine group and western medicine group increased first and then decreased,which was significantly higher than that in normal group since the 4th day(P < 0.05),and reached the peak on the 7th day(the body weight of model group was 309.00±6.44 g,the body weight of Chinese medicine group was 302.42±15.25 g,and the body weight of western medicine group was 303.96±11.61 g).Compared with the normal group(278.90±11.55g),the body weight was significantly different(P < 0.01).On the 8th day,the body weight of the three groups of rats began to decline sharply under the influence of high temperature and humidity.(2)Compared with the model group,the body weight of the Chinese medicine group began to increase on the 16 th day,and on the 21 th day,the body weight of the model group was247.82±5.89 g and that of the Chinese medicine group was 285.82±5.65 g,showing a significant difference(P < 0.01).(3)There was no significant difference in body weight between the western medicine group and the model group.3.Degree of ankle joint swelling: Before MSU crystal suspension injection,there was no ankle joint swelling in each group.Compared with the normal group,the degree of ankle swelling in the model group,the Chinese medicine group and the western medicine group increased by22.04%,23.81% and22.43%,respectively,from 8h after injection,with extremely significant differences(P < 0.01).The degree of ankle swelling in the three groups increased by 53.43%,40.48% and 27.79% at 48 h,24 h and 12 h,respectively,and the degree of swelling was the largest.Then the swelling gradually goes down.Compared with the model group,the Chinese medicine group showed significant difference from 24 h(P < 0.01),and the western medicine group showed significant difference from 12 h(P < 0.05).Compared with the western medicine group,the Chinese medicine group was significantly higher than the Western medicine group at 48 h(P < 0.05),but there was no significant difference between the two groups at 72 h(P > 0.05).4.Synovial pathology results: In the normal group,the synovial surface was smooth,the cells were single layer and neatly arranged,and less inflammatory cell infiltration was observed.In the model group,the synovial tissue structure was disordered,fibrous tissue hyperplasia was obvious,a large number of neutrophils,macrophages and other inflammatory cells were infiltrated,and pathological damage was serious.After drug intervention,compared with the model group,inflammatory cell infiltration was significantly reduced in the Chinese and western medicine groups,pathological damage was significantly reduced,no obvious fibrous tissue hyperplasia,and inflammatory infiltration was less in the western medicine group.5.The expression of NLRP3 inflammatory factor in synoviae: q PCR results showed that the m RNA expression level of NLRP3 in the model group was4.66 folds higher than that in the normal group,and the difference was extremely significant(P < 0.01).The NLRP3 m RNA expression level of Chinese and western medicine was significantly down-regulated by 62% and64%,respectively(P < 0.01),and the NLRP3 m RNA expression level was not statistically significant between the Chinese and western medicine groups.6.Results of colonic pathological changes: In the normal group,the surface of the colonic mucosa was smooth,the epithelial cells were arranged neatly and densely,the lamina propria was dense and the goblet cells were gathered,and there were no obvious pathological changes in the connective tissue of the submucosa.In the model group,the epithelial cells of mucous membrane were hyperplasia,deformation,and damage were serious.The intestinal glands of lamina propria were significantly reduced,the muscular layer atrophied,and a large number of inflammatory cells infiltrated lamina propria.Compared with the model group,the mucosa of the Chinese medicine group was smooth and inflammatory cell infiltration was significantly reduced.In the western medicine group,mucosal epithelial cells were seriously damaged,goblet cells increased and inflammatory infiltration decreased significantly.7.The expression of factors related to TLRs signaling pathway in colon tissue was as follows:(1)q PCR results: Compared with normal group,m RNA expression levels of TLR2,TLR4,My D88 and NF-κB in model group were increased by 5.26 folds,17.40 folds,23.80 folds and 4.30 folds,respectively(P< 0.01).Compared with model group,the m RNA expression levels of TLR2,TLR4,My D88 and NF-κB in Chinese medicine group were decreased by 10%,69%,75% and 60%,and that in western medicine group were decreased by26%,77%,88% and 75%,respectively(P < 0.01).Compared with western medicine group,m RNA expression levels of TLR2,My D88 and NF-κB in Chinese medicine group were significantly increased(P < 0.01).(2)WB results:Compared with the normal group,the expressions of TLR2,TLR4,My D88 and NF-κB protein in the model group were increased by 1.6 folds,1.4 folds,3.4 folds and 1.6 folds,respectively,and the differences were extremely significant(P < 0.01).Compared with model group,the expression of TLR2 protein in TCM group and western medicine group was decreased,but there was no statistical significance(P > 0.05).The expression levels of TLR4 and NF-κB protein were significantly decreased(TLR4 decreased by 77% and 84%,respectively,and NF-κB decreased by 33% and 41%,respectively)(P < 0.01).The expression level of My D88 protein was significantly decreased(18% and78%,respectively)(P < 0.05 or P < 0.01).Compared with western medicine group,the protein level of My D88 was significantly increased(P < 0.05),but the expression of other proteins was not different.Conclusions:TQF can effectively relieve the symptoms of damp-heat syndrome GA,and its mechanism is to inhibit the signaling of TLR4-My D88-NF-κB in colon and induce the regression of non-targeted inflammation in distant joints.It can be seen from the pathological morphology of colon and body weight growth that this drug is significantly better than diclofenac sodium in improving the intestinal damage caused by the disease and restoring the digestive system function of patients,which provides a theoretical basis for the clinical treatment of GA in the future. |
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