| OBJECTIVE:The compatibility relationship is formed by taking Gentianae Macrophy-llae Radix?Qinjiao?,a traditional Chinese medicine for dispelling pathogenic wind and damp-ness,as a basic medicine,and respectively matching Clematidis Radix et Rhizoma?Weiling xian?,Taxilli Herba?Sangjisheng?and Stephanlae Tetrandrae Radix?Fangji?to form a compa-tibility relationship of mild-warm?Qinjiao and Weilingxian?,mild-mild?Qinjiao and Sangji sheng?,and mild-cold?Qinjiao and Fangji?.To observe the effects of different compatibility drugs of Qinjiao on the expressions of nuclear factor kappa B?NF-?B?,vascular endothelial growth factor?VEGF?,matrix metalloproteinase-3?MMP-3?,matrix metalloproteinase tissue inhibitor-1?TIMP-1?in ankle joint of rheumatoid arthritis?RA?model rats with wind-cold-dampness arthralgia.By comparing the curative effects of Qinjiao and its different compati-bility of cold and heat,to further explore the relationship among drug properties,diseases and drug effects.METHOD:Eighty SD rats were randomly divided into blank group,disease model gro-up,wind-cold-dampness syndrome model group?Type II collagen injection plus wind-cold-dampness environmental stimulation?,positive drug group,single-taste Qinjiao group,Qinjiao-Weilingxian group?Qinwei group?,Qinjiao-Sangjisheng group?Qinsang group?,Qinjiao-Fangji Group?Qinfang group?,with 10 rats in each group.On the first day of the experiment,2points on both sides of the spinal column and 1 point on the root of the tail of the rat were shaved.Except the blank group,rats in each group were injected 0.1 mL of type?collagen emulsion intradermally at each point,totaling 0.3 mL;On the 12th day,except the blank gro-up,rats in each group were injected with 0.1 mL type?collagen emulsion into the plantar skin of the left hind foot for secondary immunization.The blank group injected the same volume of water for injection at the same time and at the same position.Starting from the next day after the initial injection of type?collagen emulsion,except for the blank group and disease model group,all other groups used the modified intelligent artificial climate box to stimulate the wind-cold-dampness environment to prepare the wind-cold-dampness arthralgia rat model combined with disease and syndrome.Wind-cold-dampness environmental conditions:temper-ature 4?,wind speed 5 m·s-1,relative humidity?95%,and rat metatarsus immersed in ice water mixture at 0?.1 time per day,30 min/per time for 18 consecutive days.After mode-ling,each Chinese medicine group was given the corresponding liquid medicine at 25 g·kg-1,the positive medicine group was given tripterygium wilfordii polyglycoside suspension at 6mg·kg-1,and the other groups were given the same amount of normal saline,once a day for 21consecutive days.During the experiment,the hair,mental state,stool,foot swelling and other general condi-tions of rats in each group were observed daily.The body mass of rats was measured and reco-rded every 3 days.The thickness of the left hind paw of rats was measured and recorded every3 days,and the swelling degree of the paw was calculated.On the 38th day of the experiment,arthritis index?AI?was scored for each group of rats.On the 39th day of the experiment,1 h after the last administration,5 mL of blood was taken from the rat's abdominal aorta.After sta-nding for 2 h,the blood was centrifuged(3000 r·min-1,15 min).The serum was taken and the contents of rheumatoid factor?RF?,C-reactive protein?CRP?,interleukin-6?IL-6?in the rat's serum were detected by ELISA kit.The left posterior ankle joints of 4 rats were taken in each group,fixed with 4%paraformaldehyde,stained with HE,and the pathological changes of the ankle joints were observed under electron microscope.The integral optical density?IOD?values of NF-?B,VEGF,MMP-3,TIMP-1 of the ankle joints of the rats in each group were detected by immunohistochemistry.The left posterior ankle joints of 3 rats were stored at-80?,and the protein expressions of NF-?B,VEGF,MMP-3 and TIMP-1 in the ankle joints of the rats in each group were detected by western blot?WB?.The left posterior ankle joints of 3rats in each group were stored at-80?,and the expressions of NF-?B mRNA,VEGF mRNA,MMP-3 mRNA and TIMP-1 mRNA in the ankle joints of each group were detected by real-time fluorescence quantitative PCR?RT-PCR?.RESULTS:1.General state observation During the experiment,the rats in the blank group were more active and their body mass increased rapidly.After secondary immunization,the activity of each model rat was significantly reduced,and the rats preferred to lie on their side.The left hind foot metatarsal swelling was obvious.The left hind foot metatarsal skin was soft and purplish red with obvious fever.Some rats showed molting phenomenon or limb swelling,and the body mass increased slowly.After treatment,the activity of rats in each administration group increased,and the swelling of the foot sole decreased to different degrees.The color of the left hind foot sole gradually returned to normal skin color from purplish red,and the body mass gradually increased.However,the disease model group and wind-cold-dampness syndrome model group did not significantly improve.2.The degree of plantar swelling and AI score Compared with the blank group,the degree of plantar swelling and AI score in disease model group and wind-cold-dampness syndrome model group were significantly higher?P<0.01?.Compared with the wind-cold-dampness syndrome model group,the swelling degree and AI score of the feet in each administration group were significantly reduced?P<0.05,P<0.01?,and Qinwei group was the most obvious one among the Chinese medicine groups.3.Histopathological results In the disease model group,the cartilage tissue structure was severely damaged,the arrangement of chondrocytes was loose,a small amount of chondrocyte nucleus shrank and died,a large amount of inflammatory cells infiltrated,a large amount of neovascularization occurred,and the articular surface was seriously damaged.On the basis of pathological changes in the disease model group,a large number of fibrous exudates and synovial cells proliferate in the joint cavity of the wind-cold-dampness syndrome model group,with some synovial cells protruding into the joint cavity as villous hyperplasia and rough joint surface.In each treatment group,the destruction of cartilage tissue structure of ankle joint was reduced,a small amount of inflammatory cells infiltrated,neovascularization was reduced,and articular surface damage was restored to different degrees.4.NF-?B expression results Compared with the blank group,the IOD value of NF-?B,NF-?B protein expression level and NF-?BmRNA level of the ankle joint in the disease model group and wind-cold-dampness syndrome model group were significantly increased?P<0.01?.Compared with the wind-cold-dampness syndrome model group,the IOD value,protein expression level and gene expression level of NF-?B in each administration group decreased to different degrees.Compared with other traditional Chinese medicine groups,the IOD value of NF-?B in ankle joint of rats in Qinwei group was significantly reduced?P<0.01?,the expression level of NF-?B protein was significantly lower than that of single-taste Qinjiao group?P<0.05?,and the mRNA level of NF-?B was significantly lower than that of Qinsang group and Qinfang group?P<0.01?.5.VEGF expression results Compared with the blank group,the IOD value of VEGF,VEGF protein expression level and VEGF mRNA level of ankle joint in disease model group and wind-cold-dampness syndrome model group were significantly increased?P<0.01?.Compared with the wind-cold-dampness syndrome model group,the IOD value,protein expression level and gene expression level of VEGF in each administration group decreased to different degrees.Compared with other traditional Chinese medicine groups,the IOD value of VEGF in ankle joint of rats in Qinwei group was significantly reduced?P<0.01?,the expression level of VEGF protein was significantly lower than that in single-taste Qinjiao group and Qinsang group?P<0.01,P<0.05?,and the level of VEGF mRNA was significantly reduced?P<0.01?.6.MMP-3 and TIMP-1 expression results Compared with the blank group,the IOD value of MMP-3,MMP-3 protein expression level and MMP-3mRNA level of the ankle joint in the disease model group and wind-cold-dampness syndrome model group were significantly increased?P<0.01?,while the IOD value of TIMP-1,TIMP-1 protein expression level and TIMP-1mRNA level of the ankle joint were significantly decreased?P<0.01?.Compared with wind-cold-dampness syndrome model group,the IOD value,protein expression level and gene expression level of MMP-3 in ankle joint of each administration group were significantly reduced?P<0.01?.The IOD value,protein expression level and gene expression level of TIMP-1 in ankle joint of rats in positive control group,Qinwei group and Qinsang group all increased?P<0.01?.The IOD value of MMP-3 in ankle joint of rats in Qinwei group was significantly decreased?P<0.01?,the expression level of MMP-3 protein was significantly lower than that in single-taste Qinjiao group?P<0.01?,and the expression level of MMP-3 mRNA was not significantly different?P>0.05?.The IOD value,TIMP-1protein expression level and TIMP-1 mRNA level of ankle joint of rats in Qinwei group were significantly increased?P<0.01?.7.RF,CRP and IL-6 results Compared with the blank group,the serum RF,CRP and IL-6 levels in the disease model group and wind-cold-dampness syndrome model group were significantly increased?P<0.01?.Compared with wind-cold-dampness syndrome model group,the levels of serum RF,CRP and IL-6 in each administration group were decreased.Compared with the groups of traditional Chinese medicine,the serum RF level of rats in Qinwei group is lower than that in single-taste Qinjiao group and Qinfang group?P<0.05?,and the serum CRP and IL-6 levels are not significantly different?P>0.05?.CONCLUSION:Qinjiao and its compatibility groups have certain therapeutic effects on wind-cold-dampness arthralgia rats.Among them,Qinwei group has the best therapeutic effect,which can significantly reduce the swelling of rat footpads,reduce apoptosis of chondrocyte nucleus and neovascularization,significantly reduce the expression of NF-?B and VEGF,down-regulate the ratio of MMP-3 to TIMP-1,and reduce cartilage destruction.The trend of curative effect of traditional Chinese medicine compatibility on wind-cold-dampness arthralgia rats is as follows:Qinwei group>Qinsang group>single-ingredient Qinjiao group>Qinfang group.The therapeutic effect of Qinwei group matched by mild and warm is better than that of Qinsang group matched by mild and mild,Qinfang group matched by mild and cold and Qinjiao group,suggesting that for cold diseases,thermal drug therapy should be given to obtain better therapeutic effect.The experimental results are basically consistent with the therapeutic principles of traditional Chinese medicine of"treating cold with thermal drug"and"treating cold with heat".The mechanism of this compatibility in treating wind-cold-dampness arthralgia type RA may be related to its ability to reduce the expression levels of NF-?B,VEGF and MMP-3,increase the expression of TIMP-1,reduce the levels of serum RF,CRP and IL-6,reduce articular cartilage damage and neovascularization. |
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