Study On The Effect And Mechanism Of Danggui Buxue Decoction On Rheumatoid Arthritis

Objective: The etiology and pathogenesis of RA are still unknown,the incidence rate is increasing year by year,and there is no specific drug at present.DBD is commonly used in clinical treatment of RA,and the curative effect is significant.Based on this,the purpose of this study is to clarify the anti-RA effect of DBD in vitro and in vivo and its potential mechanism.Methods:(1)In vivo experiment: 42 Wistar rats were divided into test and control groups at random,positive control group(Tripterygium wilfordii glycoside group),high,medium,and low dose DBD groups.In addition to the blank group,the other groups used bovine type Ⅱ collagen and Freund’s complete adjuvant to establish the CIA rat model.After 7 days of secondary immunization,the rats in the blank group and CIA group were given physiological saline for 28 days.Observe the general situation of rats,record the weight of rats,the degree of foot swelling,and score arthritis;Effect of Danggui Buxue Decoction on the mRNA expression of apoptosis markers Bax,Bcl-2,and caspase-3 in synovial tissue of rats detected by reverse transcription polymerase chain reaction;The effect of DBD on the content of IL-6 and IL-15 in synovial tissue of rats was detected by ELISA;The pathological changes of articular synovium of knee and ankle were observed by HE staining;The Bax、Bcl-2、caspase-3、p-JAK2、p-STAT1、p-STAT3 in synovium were examined by Western blot.(2)In vitro experiment: DBD containing serum was used to culture and intervene HFLS-RA.Cell counting kit-8 was used to detect the effect of DBD containing serum on the proliferation of HFLS-RA cells;The effect of DBD containing serum on apoptosis of HFLS-RA cells was detected by flow cytometry;The influence of DBD with serum on Bax,Bcl-2 and caspase-3 mRNA in HFLS-RA was studied by RT-PCR;Enzyme linked immunosorbent assay was used to detect the effect of DBD containing serum on the content of interleukin-6 and interleukin-15 in HFLS-RA cell supernatant;The influence of DBD with serum on Bcl-2 related X protein,B-cell lymphoma 2,caspase-3,rabbit anti human phosphorylated tyrosine kinase 2,rabbit anti human phosphorylation signal transduction and activation factor 1,and rabbit anti human phosphorylation signal transduction and activation factor 3 proteins in HFLS-RA cells by Western blot.Results:(1)Animal experiment: Compared with the blank control group,rats in the model group were obviously red and swollen,even deformed;Compared with the model group,the above symptoms in each administration group were significantly improved.The RT-PCR results showed that the mRNA of Bax and caspase-3 decreased significantly,but Bcl-2 mRNA was elevated(P<0.01).The Bax and caspase-3 mRNA were higher than those in the control group,but Bcl-2 mRNA was reduced(P<0.01);IL-6 and IL-15 levels were significantly higher than those in the control group(P<0.01),but IL-6 and IL-15 were significantly reduced in different dosage groups(P<0.01);It was found that the joint of knee and ankle was significantly increased,the inflammatory infiltration was evident,and the cartilage injury was evident.The results indicated that the synoviosis was more obvious,the degree of inflammation decreased,and the cartilage injury was not obvious;By Western blot,Bcl-2、p-JAK2、p-STAT1、p-STAT3 were higher than those in blank control group(P<0.01),while Bax and caspase-3 were obviously lower than those in blank control group(P<0.01).Compared with the control group,Bcl-2 、 p-JAK2 、 p-STAT1 、 p-STAT3 decreased significantly(P<0.01),while Bax and caspase-3 increased significantly(P<0.01).(2)Cell experiment: DBD-containing serum significantly inhibited the proliferation of HFLS-RA cells in a dose-and time-dependent manner(P<0.01);The proportion of apoptosis in HFLS-RA cells changed significantly after the serum containing different concentrations of DBD acted on HFLS-RA cells for 24 hours.The number of PI/Annexin-V double positive cells in HFLS-RA cells increased significantly with the increase of DBD concentration(P<0.05,P<0.01);The RT-PCR assay indicated that Bax and caspase-3mRNA in the model group were lower than blank control group,however,the level of Bcl 2mRNA was significantly increased compared with that of the control(P<0.01).In comparison to the sample group,after DBD intervention,the expression level of Bcl-2mRNA in cells decreased significantly,while the expression level of Bax and caspase-3mRNA increased significantly(P<0.01);ELISA results showed that DBD significantly reduced the levels of apoptosis-related inflammatory factors IL-6 and IL-15 in a concentration-dependent manner(P<0.01);By Western blot,Bcl-2、p-JAK2、p-STAT1、p-STAT3 in the model group were higher than those in blank control group(P<0.01),while Bax and caspase-3 were obviously lower than those in control group(P<0.01)).The results showed that Bcl-2、p-JAK2、p-STAT1、p-STAT3 in the treatment group were markedly reduced(P<0.05,P<0.01))and Bax and caspase-3 were higher than those in the model group(P<0.01).Conclusion:(1)DBD has obvious therapeutic effect on swelling and inflammation of toe joint in CIA rats,and the serum containing DBD can significantly inhibit the proliferation of fibroblast-like synovial cells and promote their apoptosis.Both in vivo and in vitro experiments show that DBD is effective in treating RA and plays a significant role in anti-arthritis.(2)The mechanism of DBD in treating RA may be related to inhibiting JAK2/STAT1/3 signal transduction,regulating the secretion of inflammatory factors and the expression of apoptosis-related factors.