The Effects Of A Series Of Prescriptions Based On Mongolian Medicine Sanzi Decoction On Joint Neovascularization In CIA Rats

Objectives1.To observe the effect of Sanzi Decoction(SZD)on synovial inflammation of CIA model rats,and compare the therapeutic effects of SZD,Sendeng 4 Decoction(SDD)and Zhonglun 5 Decoction(ZLD)in alleviating synovial inflammation.2.To clarify the effect and potential mechanism of a series of prescriptions based on Mongolian medicine SZD on the neovascularization of CIA model rats,and analyze whether SDD and ZLD have synergistic effects compared with SZD or other differences in mechanism.3.To observe the effects of a series of medicated serums based on SZD on the proliferation of RA-FLS and HUVEC in vitro,and preliminarily explore the possible mechanism.MethodsPart 1:Taking CIA rats as the research object,by evaluating their body weight,joint swelling,arthritis score(AI),joint pathological changes and the levels of inflammatory factors IL-1? and IFN-y,observe the effect of different doses of SZD,SDD and ZLD in improving the inflammation of CIA,identify the best dose,and compare the differences in the efficacy of the three groups of compound prescriptions.One hundred and twenty SD male rats were divided into normal control group(n=10)and model control group(n=110)randomly,and then prepare CIA rat models.The modeled rats were divided into the following groups randomly:model control group,TGT group(0.02g/kg per day),SZD low,medium,and high dose groups(1.35g/kg per day,2.7g/kg per day,5.4 g/kg per day),SDD low,medium and high dose groups(1.35g/kg per day,2.7g/kg per day,5.4g/kg per day),ZLD low,medium,and high-dose group(1.35g/kg per day,2.7g/kg per day,5.4 g/kg per day),6 rats in each group.Equal volumes of distilled water were given to normal control group and model control group.On the 21st day after the initial immunization,the rats were given by gavage,once a day,for 4 weeks.Observe and record the general state,body weight,and plantar swelling degree of each group rats before inflammation,administration,1 week after administration,2 weeks after administration,3 weeks after administration,and 4 weeks later,scored the AI.Before administration,blood was taken from the intraocular canthus of rats,and the content of anti-C? antibody was detected by Elisa.After 4 weeks of administration,the rats were anesthetized to collect blood and ankle joints.HE staining was used to evaluate the pathological changes in the ankle joints of rats.The expression levels of IL-1? and IFN-? in the ankle joints were detected by immunohistochemistryPart 2:Identify the effects and potential mechanisms of the three groups of compound prescriptions on improving synovial neovascularization in CIA rats,screen the optimal dose,and compare the differences of the three groups of compound prescriptions in the efficacy Continuation of experiment one,after the blood and ankle joints were collected,the levels of cytokines VEGF,eNOS,and tPA in the serum of CIA rats were detected by Elisa;the expression of VEGFA,PLGF,and HIF-? in the ankle joints were detected by immunohistochemical;Western blot was used to detect the expression of Ang-1,Ang-2 and Tie-2 in ankle joints of rats Part 3:By using serum pharmacological methods to observe the effects of three groups of compound prescriptions with different concentrations of drug-containing serum on the proliferation of RA-FLS and HUVEC in vitro,screen the optimal dose.The differences in efficacy among the three groups of compound prescriptions were compared,and combined with the detection of angiogenesis related cytokines.Prepare drug-containing serum of the three groups of compound prescriptions,establish the RA-FLS injury model induced by IL-1? and the HUVEC injury model induced by VEGF165.MTT was used to detect the cell viability of the three groups of drug-containing serum with different concentrations(5%,10%,20%)on two kinds of cell injury models in vitro.Observe the effect of three groups of compound drug-containing serum on the formation of HUVEC lumen Elisa method was used to detect the contents of VEGF,PLGF,Ang-1,Ang-2 in the supernatant of RA-FLS cells and the contents of VEGFR-1,VEGFR-2,and Tie-2 in the supernatant of HUVEC cells.ResultsPart 1:Compared with the normal control group,the content of anti-C ?antibody in serum of model rats was significantly increased(P<0.01),which indicated that CIA models were established successfully.Compared with the normal control group,The high-dose groups of SZD and ZLD could significantly increase the weight of rats(P<0.05):the high-dose groups of SDD and ZLD could significantly alleviate the joint swelling of rats(P<0.05);the medium and high-dose groups of SZD,the low,medium and high-dose groups of SDD,and the high-dose group of ZLD had a certain effect on reducing the AI of rats(P<0.05);the high-dose groups of the three groups of compound prescriptions could significantly alleviate the inflammation of ankle joint and reduce the formation of neovascularization in CIA rats(P<0.05);the high-dose groups of SDD and ZLD had obvious effect on reducing the expression of IL-1(3 and IFN-y in ankle joint of rats(P<0.05),but SZD had no obvious effect.Part 2:Elisa results showed that compared with the model control group,the high-dose groups of SZD and SDD,and the ZLD high and medium dose groups could reduce the VEGF content in the peripheral serum of rats(P<0.05);the content of eNOS in the peripheral serum of rats in each administration group was significantly reduced(P<0.05),and the high-dose groups of SZD and SDD,and the ZLD medium dose group had the best effect;the content of tPA in peripheral serum of rats in the middle and high dose groups of ZLD was significantly reduced(P<0.05).The results of immunohistochemical method showed that compared with the model control group,the high doses of SDD and ZLD had a significant effect on reducing the expression of VEGFA in the ankle joints of rats(P<0.01);the medium and high doses of SZD,SDD and ZLD all had a decreasing trend in the expression of PLGF in the ankle joints of rats,but the overall difference was not significant(P>0.05);the high doses of SDD and ZLD could significantly reduce the expression of HIF-? in the ankle joints of rats(P<0.05).Western blot results showed that compared with the model control group,The high doses of SZD,ZLD and the medium and high doses of SDD had a tendency to down-regulate the expression of Ang-1 protein in the ankle joint of rats,but there was no statistical significance(P>0.05);the expression of Ang-2 in the ankle joints of rats in each dose group was significantly reduced(P<0.05);the medium dose groups of SZD and SDD could significantly reduce the expression of Tie-2(P<0.05).Part 3:MTT results showed that compared with IL-1? induction group,the three groups of drug-containing serum all had significant effects in inhibiting the proliferation of RA-FLS(P<0.05),and the optimal concentration was 20%;compared with VEGF165 induction group,the three groups of compound drug-containing serum could significantly inhibit the proliferation of HUVEC(P<0.05),and the 20%concentration of SZD,10%and 20%of ZLD drug-containing serum had the most significant effect;the experiment of the two kinds of cells showed that the best intervention time of the drug was 24 hours.The results of HUVEC lumen formation experiment showed that the drug-containing serum of the three groups of 20%concentration could reduce the lumen formation of HUVEC induced by VEGF 165,but there was no significant difference(P>0.05).The results of Elisa showed that compared with IL-1?induction group,10%,20%SZD,SDD and ZLD could reduce the content of VEGF(P<0.01);each concentration group of the three groups of compound drug-containing serum could reduce the contents of PLGF and Ang-1(P<0.01);20%SZD,10%,20%SDD and ZLD could significantly reduce the content of Ang-2(P<0.05);compared with VEGF165 induction group,each concentration group of the three groups of compound drug-containing serum could reduce the contents of VEGFR-1(P<0.01);10%,20%SZD,5%,10%,20%SDD and ZLD could reduce the content of VEGFR-2(P<0.01);10%,20%SZD,SDD and ZLD could reduce the content of Tie-2(P<0.05);the optimal concentration of the above drug-containing was 20%.ConclusionsSDD and ZLD had a certain synergistic effect in relieving RA joint inflammation compared with the basic prescription SZD,but there was no significant difference between the two prescriptions.The three groups of compound prescriptions could alleviate CIA joint neovascularization by regulating the key cytokines involved in the HIF-VEGF-Ang,but the mechanism of their pharmacological effects was biased.SZD mainly worked by reducing the expression of VEGF and eNOS and inhibiting the Ang-2/Tie-2 pathway;SDD worked by reducing the expression of VEGF,HIF-?,eNOS and inhibiting the Ang-2/Tie-2 pathway;ZLD worked by down-regulating the expression of VEGF,HIF-?,eNOS and Ang-2.SDD had more targets and a stronger anti-angiogenesis effect.It provided scientific basis for the treatment of RA with a series of prescriptions based on SZD,and for further research on the optimized prescription of Mongolian medicine and the development of new drugs.