Research On The Processing Crafts Of Daphne Giraldii Nitsche With Liquorice And Its Anti-rheumatoid Arthritis’ Mechanism

Objective:In this study,the content of daphnetin was used as the evaluation index,the response surface methodology was used to investigate the licorice-fried and licorice-baked technology of Daphne giraldii Nitsche.Therapeutic effects and mechanism of raw Daphne giraldii Nitsche and two kinds of licorice-processed Daphne giraldii Nitsche products in rheumatoid arthritis treatment were evaluated both in type II bovine collagen-induced rheumatoid arthritis rats model and an LPS-induced Raw264.7 cell inflammation.Methods:1.The effects of ratio of licorice juice and Daphne giraldii Nitsche,moistening time,frying temperature and frying time on processing technology of licorice-fried Daphne giraldii Nitsche were investigated with the content of daphnetin as the evaluation index by using response surface test with three factors and three levels.2.The effects of ratio of licorice juice and Daphne giraldii Nitsche,moistening time,baking temperature and baking time on processing technology of licorice-baked Daphne giraldii Nitsche were investigated with the content of daphnetin as the evaluation index by using response surface test with three factors and three levels.3.Collagen-induced rheumatoid arthritis(CIA)rat model was established and the therapeutic effect of raw Daphne giraldii Nitsche and licorice-processed Daphne giraldii Nitsche products on CIA rats was evaluated by weight,arthritis score,foot swelling degree,spleen index,thymus index,histopathology evaluation and X-Ray radiological observation of inflamed ankle joints.4.The effect of raw Daphne giraldii Nitsche and licorice-processed Daphne giraldii Nitsche products on the expression of tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-1β,IL-10,matrix metalloproteinase(MMP)-1,MMP-3,and vascular endothelial growth factor(VEGF)in serum were measured by enzyme linked immunosorbent assay(ELISA).Expression quantity of NOD like receptor family pyrin domain-containing 3(NLRP3)and nuclear factor kappa B(NF-κB)evaluated with immunohistochemistry in ankle joint of CIA rats.Moreover,the molecular mechanism of raw Daphne giraldii Nitsche and licorice-processed Daphne giraldii Nitsche products was elucidated by using Western blot through the analysis of protein and levels of Toll-like receptor4(TLR4),NLRP3,NF-κB,IL-1β in synovia of rheumatoid arthritis rats.5.Induced by lipopolysaccharide(LPS)in Raw264.7 cells for the model,the cell proliferation and cell toxicity detection kit(CCK8)microplate method to determine cell proliferation viability and NO content in the cell,enzyme-linked immunoassay(ELISA)in cell supernatant on TNF-α,IL-6,IL-1β,IL-10,and Western Blot method was developed for the determination of cell TLR4,NLRP3,NF-κ B,IL-1β expression,preliminary validation the anti-inflammatory action mechanism of raw Daphne giraldii Nitsche and licorice-processed Daphne giraldii Nitsche products.Results:1.The optimal processing technology for frying Daphne giraldii Nitsche with licorice was as follows: the ratio of licorice juice to Daphne giraldii Nitsche was 0.8,the moistening time was 53 min,the frying temperature was 150℃,and the frying time was14 min.2.The optimal processing technology of baking Daphne giraldii Nitsche from licorice was as follows: the ratio of licorice juice to Daphne giraldii Nitsche was 0.8,the moistening time was 62 min,the drying temperature was 150℃,and the drying time was100 min.3.The results of pharmacodynamic showed that licorice-processed Daphne giraldii Nitsche lowered the foot swelling degree and the arthritis score of CIA rats,relieved the redness and swelling of their ankles and hindfeet,and significantly alleviate pathological injuries of ankle and articular cartilage in CIA rats(P<0.05).There were no significant differences between rats in the licorice-fried Daphne giraldii Nitsche products and licorice-baked Daphne giraldii Nitsche products and those in the RDGN group,but the former presented a more significant trend of ankle lesion improvement.4.The mechanism of licorice-processed Daphne giraldii Nitsche products in the treatment of rheumatoid arthritis in rats showed that they could reduce pro-inflammatory cytokines(TNF-α,IL-6 and IL-1β),increase the anti-inflammatory cytokine IL-10,and reduce MMP-1,MMP-3 and VEGF both in vivo(P<0.05).In addition,licorice-processed Daphne giraldii Nitsche products were possible to treat rheumatoid arthritis by inhibiting the activation of TLR4/NF-κB/NLRP3 pathway to play an anti-inflammatory role in treating rheumatoid arthritis.5.In vitro results showed that Daphne giraldii Nitsche could regulate the expression of NO and inflammatory cytokines TNF-α,IL-6,IL-1β and IL-10 in the supernatant of LPS-induced RAW264.7 cells(P<0.05),and the mechanism of action might be related to the inhibition of TLR4/NF-κB/NLRP3 pathway.Conclusion:In this study,response surface methodology was used to optimize the processing parameters of fried and baked Daphne giraldii Nitsche from licorice.Pharmacodynamics results showed that different processed products of Daphne giraldii Nitsche could relieve the symptoms of rheumatoid arthritis in rats,and the therapeutic effect of processed products of Daphne giraldii Nitsche was better than that of raw Daphne giraldii Nitsche.In vivo and in vitro analysis of its mechanism of action,it was concluded that Daphne giraldii Nitsche alleviated rheumatoid arthritis symptoms and inhibited RAW264.7 cell inflammation in CIA rats,which may be related to the regulation of inflammatory factors and inhibition of TLR4/NF-κB/NLRP3 pathway.