| OBJECTIVE To simulate the causes of convulsions in humans and establish a rat model of rheumatic fever and rheumatic fever combined with syndromes and syndromes in accordance with the characteristics of traditional Chinese medicine syndromes.Through the effects of different combinations of cold and heat on the compatibility of Gentiana and Radix,the intervening effects of Gentiana macrophylla and its medicinal herbs on RA rats with rheumatic fever were studied from the perspective of molecular biology,and the differences between cold,heat,and level of different drug compatibility and rheumatic fever syndrome were analyzed.Relationships to explore the relationship between drug and disease,drug and efficacy.METHOD1.Establishment of rheumatic fever RA rat model After 1 week of adaptive feeding,eighty SD rats were random Ly divided into blank group,collagenⅡmodel group,disease syndrome model group（collagenⅡ+rheumatic fever）,positive drug group（tripterygium glycosides tablets）,Qin-Wei group（flat+warm）,Qin-Sang group（flat+flat）,Qin-Fang group（flat+cold）,10 per group,a total of 8 groups.On the 1st day of the experiment,except for the blank group,the rest of the rats in each group took 2 points on the back（both sides of the spine）and 1 at the base of the tail,shaved,and injected subcutaneously with 0.1 m L of CII emulsion,0.3 mL in total.On the12th day of the experiment,0.1 mL CII emulsion was injected intradermally into the left hind paw of the rat for secondary immunization.The blank group was injected with the same amount of saline at the same site.From the 2nd day after the first injection of the CII emulsion,except for the blank group and the type II collagen group,all the other groups used the modified artificial climate box to model the rheumatic fever syndrome model.The simulated conditions for rheumatic fever were as follows:temperature 3638°C,wind speed 5 m/s,relative humidity≧95%.The experiment was steam fumigation at 37°C once a day for 30 minutes each time（including air exchange every 10 minutes）,for 11 consecutive days.On the 12th day of the experiment,rats were injected subcutaneously with 0.1 mL of CII emulsion in the left hind paw and then injected into artificial climate chambers.The environmental conditions were unchanged,and the rheumatic environment was stimulated continuously for 7 days.The total duration of co-modeling was 18 days.2.Administration and general observation After the completion of modeling,each Chinese medicine group was given intragastricly at a dose of 25.05 g·kg-1 and Tripterygium glycoside tablets at 6 mg·kg-1.The blank group,type II collagen group,and syndrome model group were given the same amount of physiology.Saline,once daily for 21 days.In the experiment,the body weight and the degree of swelling of the ankle joints were measured every 3 days for each group of rats.The mental status,drinking water,urine,color of the ankle and foot area,and changes in weight were observed daily.3.X-ray film On the 35th day of the experiment,live double-hinge x-ray radiographs were performed on each group of rats using a multi-functional living-body imager（KODAK）and interpreted.4.Histopathological observation On the 40th day of the experiment,animals were sacrificed,and 2 left hind limb ankle joints were taken from each group,fixed in4%paraformaldehyde solution,decalcified,embedded in paraffin,sectioned and stained with HE.The morphological changes of joint tissues were observed under light microscope.5.Immunohistochemical detection Rats were dehydrated by pathological section of the sacroiliac joints,and the neutrophil was fixed in a transparent gel and microscopically observed.The cytoplasm was stained with yellowish-brown to brownish-yellow granular cells.The IPP 6.0 was used to measure optical density values of VEGF,NF-κB,MMP-1 and TIMP-1 dyed into brown-yellow particles in the sacroiliac joints.6.Protein expression levels The protein expression of VEGF,NF-κB,MMP-1,and TIMP-1 in the ankle joints of rats in each group was semi-quantitatively analyzed by Western blot technique.The gray value processing and data of the bands were performed by ImageJ and SPSS 21.0 software analysis.7.Gene expression level studies Quantitative detection of VEGF mRNA,NF-κB mRNA,MMP-1 mRNA and TIMP-1 mRNA expression in the ankle by using real-time fluorescence quantitative polymerase chain reaction（RT-PCR）was performed.SPSS 21.0 software was used for data analysis and processing.RESULTS1.General condition observation From the general state of the rats,the rats of blank group had high activity,good mental state,and shiny hair throughout the experiment.Rats in the modeling group showed different states at different stages:In the primary immunization stage,rats had less activity in each model group,hair loosen,diet water reduced,and the left and right hind paws all showed different degrees of redness,but the degree of Lighter,does not affect activities.Severe swelling of the foot after the second booster immunization.The left foot is dragged or lifted.It cannot bear weight.The swollen part is warm and is rejected.The skin color changes from tender red to deep red,and finally turns dark red.The touch is from soft to stiff.The number of battles in rats increased,irritable and aggressive.Dietary drinking water is significantly reduced,individual urine is golden yellow.After drug treatment,left and right hind paws of rats in each administration group had different degrees of regression of fever,and the mental state improved,the degree of activity increased,and the drinking water gradually became normal.However,there was no obvious improement between the type II collagen group and the disease model group.2.Imaging results The results of X-ray showed that the bilateral sacroiliac joint space was clear in the blank group.the articular surface was smooth.the bone structure was normal and the normal sacroiliac joint performance was observed.However,bilateral soft tissue swelling around the joints and narrowing of joint space were observed in the type II collagen group and the syndrome model group rats.The joint space narrowing in the model group was significantly higher than that in the type II collagen group.the joint space in each group was observed.The stenosis was improved and the bone erosion lesions were still clearly visible.The soft tissue swelling around the bilateral joints of the rats disappeared in Qin-fang group,which the stenosis of the joint space was most obviously improved.3.Histopathological results Histopathological results showed that the articular surface of collagenⅡand disease syndrome model were completely destroyed.The trabecular bone became thinner.The medullary cavity widened.Broaden the medullary cavity,a large number of inflammatory cell infiltration,a large number of granulation tissue hyperplasia,peripheral vascular rich and serious expansion,angiogenesis,fibrosis.The damage of the rats of disease and syndrome model was significantly higher than the collagenⅡ.Although the articular surface in varying degrees of destruction,less inflammatory cells,granulation tissue aging,collagen fiber hyperplasia,fibrosis increased scarring,articular surface of Qin-Fang group repair the best.4.VEGF expression results Compared with the blank group,the integrated optical density,relative protein expression and gene expression levels of VEGF was increased to varying degrees in the sacroiliac joint of type II collagen group and syndrome model group,and the disease syndrome model group was significantly higher than II collagen group（P<0.01）.Compared with the syndrome model group,the integrated optical density,relative protein expression,and gene expression levels of VEGF decreased in different degrees in each administration group.Qin-fang group was significantly lower than other groups（P<0.01）.5.NF-κB expression results Compared with the blank group,the integrated optical density,relative protein expression and gene expression levels of NF-κB was increased to varying degrees in the ankle joint of type II collagen group and disease model group,and the disease model group was higher than type II collagen group（P<0.05）.Compared with the syndrome model group,the integrated optical density,relative protein expression,and gene expression levels of NF-κB were decreased in different dose groups（P<0.01）.Compared with each group,the integrated optical density and relative protein expression of NF-κB in Qin-fang group was decreased most significantly（P<0.05）,but there was no significant difference in gene expression level（P>0.05）.6.MMP-1 and TIMP-1expression results Compared with the blank group,the relative expression levels of MMP-1 protein and gene expression levels in the sacroiliac joint of type II collagen group and disease model group was increased to varying degrees,but TIMP-1 was decreased.MMP-1 was Significantly higher in disease syndrome model group than type II collagen group（P<0.05）,but TIMP-1 was lower than type II collagen group（P<0.05）.Compared with disease model group,relative expression level and gene expression of MMP-1 protein had different degrees of reduction（P<0.05）in each dose group,and TIMP-1 was increased to varying degrees（P<0.05）.The relative expression of MMP-1 protein in Qin-fang group was significantly lower than that in other groups（P<0.05）.TIMP-1 was higher than other groups（P<0.05）,but there was no significant difference in the expression level of MMP-1 gene（P>0.05）.TIMP-1 gene expression was significantly higher than other groups（P<0.05）.CONCLUSIONThis study was based on the effects of different combinations of cold and heat on the rheumatic fever RA,through X-ray film,histopathological observation,immunohistochemical detection,protein expression levels,gene expression levels,etc.The results showed that:1.Based on the comprehensive experimental results,it was confirmed that the expression levels of VEGF,NF-κB,MMP-1 and the mRNA expression levels were positively correlated with the degree of inflammation in the RA ankle joint tissue,while the expression level and mRNA and protein of TIMP-1 is inversely related to the degree of inflammation.The expression of VEGF,NF-κB,MMP-1 the expression of TIMP-1 were increased in the rat model group,which indicated that the normal dynamic balance expression of the key molecules was broken,and the molecular regulation mechanism in the body was changed,which aggravated the condition.2.The effects of different combinations of cold and heat on the treatment of rheumatic fever RA may be related to the decrease of expression VEGF,NF-κB and MMP-1,increase expression of TIMP-1,decrease inflammatory cell infiltration,reduction vasospasm,and reduction articular cartilage destruction.3.Treatment of rheumatic fever RA rats and Qin-Fang group（flat+cold）could significantly reduce the expression and mRNA expression of VEGF,NF-κB,MMP-1,increase the expression of TIMP-1 and mRNA expression.In order to reduce the degree of inflammation,relieve cartilage damage the effect is more prominent,which reflect the principle of Chinese medicine treatment of"warm cold"and"treating cold with cold medicine". |
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