| OBJECTIVE:To investigate the anti-inflammatory effects and potentiation mechanism of Gentiana macrophylla,Chinese Clematis and Gentiana macrophylla-Chinese Clematis couplet medicines on rheumatoid arthritis（RA）model rats through the intervention administration of Gentiana macrophylla,Chinese Clematis and their compatibility decoction.METHODS:After 3 days of adaptive feeding,48 SD rats were divided into 6 groups according to the random number table method,including blank group,model group,positive drug（methotrexate）group,single Gentiana macrophylla group,single Chinese Clematis group,and Qinwei compatibility group（Gentiana macrophylla combined with Chinese Clematis group）,with a total of 8 rats in each group.Except for the blank group,the RA model was replicated in all groups.After successful modeling,the positive drug group was given methotrexate solution at 0.79 mg·kg-1by gavage twice a week;the single Gentiana macrophylla group,the single Chinese Clematis group and the Qinwei compatibility group were given the corresponding water decoction at 21 g·kg-1by gavage,and the blank group and the model group were given the corresponding volume of saline by gavage once a day for 15days.During the experiment,the general status of each group of rats was observed every day,and the body mass,right hind foot and plantar thickness and foot and plantar swelling were measured every 3 days,and the arthritis index（AI）was scored at different stages of modeling and drug administration;After drug administration and material collection,the immune organ index of each group of rats was measured and calculated;the histopathological observation of the ankle joint of each group of rats was carried out;the enzyme linked immunosorbent assay（ELISA）was used to detect the arthritis in each group of rats.The levels of rheumatoid factor（RF）,anti-cyclic citrullinated peptide（CCP）antibody,C-reactive protein（CRP）,prostaglandin E2（PGE2）,B-cell lymphoma-2（Bcl-2）,acetylcholinesterase（ACh E）and butyrylcholinesterase（BCh E）in the ankle joints of rats in each group were measured by ELISA;immunohistochemical techniques were used to detect The mean optical density（AOD）values of hypoxia-inducible factor-1α（HIF-1α）and vascular endothelial growth factor（VEGF）were measured by immunohistochemistry;the relative protein expressions of HIF-1αand VEGF in the ankle joints of rats were measured by western blot;real-time fluorescence quantitative PCR（RT-PCR）was used to measure the relative protein expressions of HIF-1αand VEGF in the ankle joints of rats.The relative gene expression of HIF-1αand VEGF in the ankle joint of each group of rats was measured by real-time fluorescence quantitative PCR（RT-PCR）.Results.1.General condition observation The rats in the blank group were in good mental condition,active,with dense,soft and shiny fur,and no changes were seen in the foot and plantar.The rats of each model group were irritable and irritable,with fluffy and yellow hair,obvious swelling of the feet,tight skin of the feet,purple or dark red in color,and fond of gathering.After treatment,compared with the model group rats,the mental state,hair softness,swelling of foot and plantar area and skin color of foot and plantar area of each group showed different degrees of improvement,and the positive drug group and Qinwei compatibility group had the best improvement effect;the model group rats did not show any obvious changes.2.Results of foot-plantar swelling After the first immunization,no significant changes were observed in the swelling of the foot and plantar area in each group;compared with the blank group,the swelling of the foot and plantar area in all groups of the modeling rats increased after the second booster immunization（day 9 of the experiment）,with highly significant differences（P<0.01）.On the 13th day of the experiment,the swelling of the foot and plantar area of the rats in the modeling group reached the highest level;on the 14th day of the experiment,the administration of the drug began,and after the administration of the treatment,the swelling of the foot and plantar area of the rats in each administration group decreased to different degrees,and on the 18th day of the experiment,the swelling of the foot and plantar area of the rats in each administration group decreased compared with that in the modeling group,and there was a highly significant difference（P<0.01）,among which the reduction effect was most significant in the Qin Wei compatibility group.3.AI score results On the 14th day of administration,compared with the model group,the AI scores of all dosing groups were reduced with highly significant differences（P<0.01）,with the most significant reduction in the Qin Wei compatibility group.4.Results of immune organ indices Compared with the blank group,the thymus indices of rats in the model group decreased with highly significant differences（P<0.01）;compared with the model group,the thymus indices of rats in the positive drug group increased with significant differences（P<0.05）.There was no statistically significant difference between the spleen index of rats in each group before and after modeling（P>0.05）.5.Histopathological results Compared with the blank group,the joint tissue structure of the rats in each modeling group was damaged to different degrees and inflammatory cell infiltration was present.Compared with the model group,the destruction of joint tissues of rats in each administered group was improved,inflammatory cells were reduced,and the cell status was improved to different degrees,among which the improvement was obvious in the positive drug group and the Qin Wei compatibility group,and more inflammatory cells infiltrated still existed in the single Gentiana macrophylla group and the single Chinese Clematis group.6.ELISA results Compared with the blank group,the serum RF,anti-CCP antibody,CRP,PGE2,Bcl-2,ACh E and BCh E contents of the model rats were significantly different（P<0.01）.Compared with the model group,serum RF,anti-CCP antibody,CRP,PGE2and Bcl-2 showed different degrees of decrease and ACh E and BCh E showed different degrees of increase in each drug administration group,among which the positive drug group had the most significant effect on the decrease of CRP and Bcl-2 content in serum（P<0.01）and the most prominent trend of increasing ACh E content in serum（P<0.01）;the Qin Wei compatibility group had the most significant effect on the decrease of serum RF,anti-CCP antibody and PGE2level（P<0.01）and the most significant effect on the increase of serum BCh E level（P<0.01）.7.The results of immunohistochemical techniques Compared with the blank group,the AOD values of HIF-1αand VEGF in the ankle joint of rats in the model group were significantly increased（P<0.01）;compared with the model group,the AOD values of HIF-1αand VEGF in the ankle joint of rats in each administration group were significantly decreased with significant differences（P<0.05,P<0.01）,with the most significant decrease in the positive drug group.8.Western Blot assay results The protein expression levels of HIF-1αand VEGF in the ankle joint of rats in the model group were significantly increased compared with the blank group（P<0.01）.After drug administration,the protein expression of HIF-1αand VEGF in the ankle joint of rats in each drug administration group was significantly reduced compared with the model group（P<0.01）,with the most significant reduction in the positive drug group.9.RT-PCR test results The expression levels of HIF-1αm RNA and VEGF m RNA in the ankle joint of rats in the model group were significantly higher compared with the blank group（P<0.01）.After drug administration,compared with the model group,the expression levels of HIF-1αm RNA,VEGF m RNA in the ankle joint of rats in each drug administration group were reduced to different degrees,among which the reduction effect was the strongest in the Qinwei compatibility group（P<0.01）.ConclusionGentiana macrophylla,Chinese Clematis and Gentiana macrophylla-Chinese Clematis couplet medicines could effectively improve the general status of RA model rats,regulate the level of serum inflammatory factors and inhibit the expression of HIF-1α,VEGF protein and gene in the ankle joint of RA model rats.And among the herbal administration groups,the Qinwei compatibility group was effective in reducing the expression of serum RF,anti-CCP antibody,CRP,PGE2,Bcl-2 and elevating ACh E and BCh E in rats,and inhibiting the expression of HIF-1α,VEGF protein and gene in the ankle joint of rats,indicating that the combination of the two drugs has synergistic effect in the treatment of RA.The mechanism of action may be through improving the expression level of inflammatory factors in serum,inhibiting the expression of HIF-1αand VEGF in ankle joint,and reducing joint vascular neovascularization and synovial vascular opacification formation,so as to achieve the purpose of treating RA. |
