| Part One Therapeutic effect of baicalein on collagen-induced arthritis miceOBJECTIVE: This part aim to observe the effect of baicalein(BAI)on collagen-induced arthritis(CIA)mice and explore possible mechanisms.METHODS:(1)The CIA model was established by DBA/1 mice with bovine type II collagen.(2)After the second immunization,grouped into four group(CON,CIA,DMSO,BAI).After the second immunization,the drug was administered intraperitoneally every other day with 50 mg/kg,and the DMSO group was intraperitoneally injected with the same amount of DMSO.(3)The arthritis score was recorded every other day.(4)After recording the 3-week arthritis score,the X-ray of the ankle joints of each group of mice was performed when the arthritis score of the CIA group reached the highest level.(5)The peripheral blood of each group was taken before mice were sacrificed.The expression of TNF-?,IL-1? and IL6 in each group was detected by ELISA.(6)The ankle joints of each group were removed and fixed with 4% paraformaldehyde.The paraffin sections were decalcified and H&E staining was used to observe the degree of ankle joint lesion.RESULTS:(1)After secondary immunization,CIA model was successfully established.(2)Arthritis score results: The arthritis score of the BAI was higher than that of the CON,and lower than that of the CIA and the DMSO.There was no significant difference in arthritis scores between the DMSO and the CIA.(3)ELISA results: the amount of TNF-? in peripheral blood of each was CON(326.1±49.68 pg/ml),CIA(525.8±21.68 pg/ml),DMSO(448.1±37.08pg/ml),BAI(299.9±72.02 pg/ml);IL-? amount was CON(46.68 ± 3.058 pg/ml),CIA(127.3±1.101pg/ml),DMSO(74.12±1.901pg/ml)),BAI(48.37 ± 0.9334 pg / ml);IL-6 amount,CON(78.13 ± 4.259 pg / ml),CIA(194 ± 1.58 pg / ml),DMSO(117.4 ± 2.836 pg / ml),BAI(82.03±2.754 pg/ml).the inflammatory factors of CIA were significantly higher than CON,DMSO decreased slightly,BAI significantly decreased.(4)X-ray: Compared with the CON and the BAI,the X-ray of the ankle joint of the CIA and the DMSO showed obvious soft tissue swelling and joint destruction.(5)H&E: compared with the CON,the adipose tissue in the subchondral bone was replaced by inflammatory infiltration.The synovial hyperplasia was observed in the CIA,the DMSO and the BAI.The degree of hyperplasia in the BAI was lower than that in the CIA and the DMSO.CONCLUSION:(1)BAI has protective effect on CIA mice,which can alleviate swelling,synovial hyperplasia and bone destruction of ankle joint.(2)BAI can reduce the expression of TNF-?,IL-1? and IL6 in peripheral blood of CIA mice.Part Two Effect of baicalein on proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis and the possible mechanismOBJECTIVE:(1)This part aim to detect the proliferation of soft tissue in the ankle joint of CIA lesions and its possible mechanism.(2)To observe the effect of baicalein on the proliferation of fibroblast-like synoviocytes(FLS)in patients with rheumatoid arthritis(RA).(3)To observe the effect of BAI on apoptosis in RA FLS and explore the possible mechanism.METHODS:(1)The expression of PCNA,AKT and P-AKT in the soft tissue around the ankle joint of each group of mice in the first part was detected by WB.(2)FLS were isolated from RA patients in vitro.CCK8 were used to detect the effects of different concentrations(5?M,10?M,20?M,40?M,80?M)on the proliferation of FLS.(3)Annexin V kit staining : The effects of different concentrations(5?M,10?M,20?M,40?M,80?M,160?M,320?M)BAI on FLS apoptosis were detected by flow cytometry.(4)RA FLS were cultured with BAI at a concentration of 160 ?M.Tunel staining was used to investigate the effect of BAI on FLS apoptosis.WB was used to detect the expression of AIF,BCL-2,BAX,Caspase3 and active Caspase3.RESULTS:(1)WB experiment found that PCNA expression increased in CIA group,DMSO group and BAI group compared with CON group,PCNA expression in BAI group was lower than CIA and DMSO group.Three experiments were repeated and grayscale analysis revealed statistically significant differences.There was no significant change in AKT in the 4 groups,and the trend of P-AKT was consistent with the PCNA trend,and the difference was statistically significant.(2)FLS was successfully cultured form 6 RA patients.(3)CCK8 results: There was no significant difference in OD values between CON group and DMSO group.The OD value was decreased by BAI,and there was no significant difference in OD values between 5 ?M and 10 ?M;there was no statistically significant difference between 20 ?M,40 ?M and 80 ?M.(4)Annexin V flow detection of apoptosis revealed a significant increase in apoptosis after 48 hours of culture in medium containing 160 ?M and 320 ?M BAI,compared with DMSO group(2.98%),160 ?M group(12.23%)and 320 ?M group(36.8%)apoptosis.The rate has risen significantly.(5)The WB experiment found that BCL-2 decreased and active caspase3 increased significantly in the BAI group(160 ?M)compared with the DMSO group.CONCLIUSION:(1)1BAI can inhibit the proliferation of RA FLS,and the inhibitory effect increases with the increase of concentration.It also has the ability to inhibit proliferation in vitro.(2)BAI can induce apoptosis of RA FLS at higher concentrations.(3)BAI mainly causes the expression of active caspase3 to increase by decreasing the expression of BCL-2,and finally causes apoptosis.Part Three Effect and mechanism of baicalein inhibiting TLR4 receptor pathway expression in fibroblast-like synoviocytes of rheumatoid arthritisOBJECTIVE:(1)This part aim to observe the effect and mechanism of BAI on the expression of inflammation induced by TLR4 activation in RA FLS.(2)Observe the effects of BAI on TLR4,TRAF6 and P65 in vivo.METHODS:(1)RA FLSs were stimulated with LPS at different concentrations and different times.The changes of IL-6 were detected by PCR,and the appropriate concentration and time point were selected.(2)After using different concentrations of BAI to act on FLS,LPS was added to RA FLS.Than the changes of IL-6 were detected by PCR.(3)RA FLS were treated with inhibitors of P65,P38,ERK,and JNK,and RA FLS was stimulated by LPS,and the changes of IL-6 were detected by PCR.(4)RA FLS were stimulated with LPS at different time,and WB was used to detect the expression of P65,P38 and its phosphorylation.(5)After treated by BAI,RA FLS was stimulated by LPS,and WB was used to detect the expression of TLR4,My D88,TRAF6,P65,P-65,P38,P-P38,ERK,P-ERK,JNK,and P-JNK.(6)The expression of TLR4 in the soft tissue of the mouse was detected by WB.RESULTS:(1)LPS significantly up-regulated the expression of IL-6 in RA FLS,and stimulation with 1 mg/m L for 12 hour was better.(2)BAI can inhibit the expression of IL-6 induced by LPS.(3)The expression of TNF-? and IL-6 was decreased after P65,P38 or ERK inhibition.JNK has no significant inhibitory effect on LPS-induced IL-6.(4)Phosphorylation of P65 and P38 was significantly increased after LPS addition,and decreased rapidly within 3 hours,and P-P65 and P-P38 expression was higher at 30 minutes.(5)BAI can specifically inhibit the phosphorylation of P65,and the inhibitory effect is more obvious with increasing concentration.At the same time,BAI can inhibit the expression of TLR4 and TRAF6.(6)BAI inhibited the expression of TLR4,TRAF6,P-P65 in vivo.CONCLUSION:(1)LPS can significantly induce the expression of IL-6 in RA FLS.(2)BAI can reduce LPS-induced expression of RA FLS inflammation by inhibiting the TLR4-TRAF6-NF-k B pathway.(3)In vitro,BAI can reduce the expression of TLR4 and TRAF6,inhibit the phosphorylation of downstream P65,resulting in decreased inflammation. |
PDF Full Text Request