| Objective:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by the primary pathologic expression of joint hypertropism.The etiology and pathogenesis of the disease are unknown,but it is currently thought that the excessive proliferation of Th17 cells is an important pathogenesis of rheumatoid arthritis.Zanthoxylum nitidum(Roxb.)DC.is the dry root of rutaceae Zanthoxylum bungeanum genus,with the function of promoting blood circulation to remove blood stasis,promoting qi circulation and relieving pain,dispelling wind and removing obstruction in the meridians,detoxicating detumescence,and the major functions are traumatic injuries,a toothache,neuralgia and rheumatism palsy etc.Nitidine chloride(NC)is the main active alkaloids of Zanthoxylum nitidum,modern pharmacological experiments prove that it has the functions of anti-inflammatory,analgesic andantitumor etc.This experiment examines the inhibition of the Zanthoxylum nitidum of the cell in the CIA mouse on Th17 cell proliferation,and provides the basis for the treatment of rheumatoid arthritis.Method:The DBA/1 mice,SPF,male,6 to 7 weeks,weight(20±2)g,with chicken type II collagen after complete emulsified with freund’s complete adjuvant,mice tail root in each experiment,back more intradermal injection of 0.1 ml hybrid emulsion.After 21 days,using the same method to strengthen the immune 1 times.Arthritis index score(AI)4 points or more means building successful.Then prepare the suspension of spleen lymphocyte cells,induced by chicken type II collagen in vitro CIA mice spleen lymphocyte proliferation,inspecting the factors of whether the collagen is inactivated and IL-23 influence effects on Th17cell proliferation in vitro model.The inhibition rate of the normal mouse splenic lymphocyte was measured by cck8,the inhibition rate of the normal mouse splenic lymphocyte was measured,and the appropriate concentration of the drug was investigated.CIA mice spleen lymphocytes can be divided into the methotrexate group and nitidine chloride group with high,medium and low concentration.Add methotrexate 0.4μg/ml or nitidine chloride 2μg/ml,1μg/ml,0.5μg/ml culturing together.After cultured 60h in vitro,using flow cytometry detected the ratios of each group of CD4~+RORγt~+Th17 cells.Results:1.The influence factors of the extracorporal proliferation model of Th17cells were investigatedBefore culturing,the ratio of the normal mice and the CIA mouse splenic lymphocyte Th17 cells(%)was 0.333±0.153,0.933±0.208respectively.Culturing after 60h,normal control group,normal collagen with not inactivated stimulus,normal collagen with inactivated stimulation group Th17 cell ratio(%)were 0.067±0.056,0.1±0.1,0.067±0.056.The control group of the CIA,the CIA with activated collagen stimulus group,the CIA with inactivated collagen stimulus group developed 60h and then the Th17 cell ratio(%)was 0.533±0.058,1.83±0.5,1.95±0.265respectively.The ratio of Th17 cells of CIA with not inactivated collagen stimulation group and the CIA with inactivated collagen stimulation group rose sharply after the culturing,compared with the CIA in the control group was statistically significant difference(p<0.01),and the inactivated stimulate collagen compare with the activated collagen stimulation group has no statistical difference(p=0.193).IL-23 of Th17 cells in vitro proliferation model results showed that the influence of culture,after 60h,the ratio of Th17 cells of blank control group,IL-23 in high dose group,IL-23 in medium dose group,IL-23 in low dose group,IL-23 p19 antibody neutralization were 1.73±0.58,1.70±0.17,1.67±0.06,1.87±0.15,0.57±0.06 respectively.IL-23 in high,medium and low dose group compared with blank control group had no significant difference,but the ratio of Th17 cells in IL-23 p19 antibodies neutralization group was significantly decreased.Compared with the blank control group,it had statistically significant difference(p<0.01).2.Nitidine chloride on Th17 cell proliferation inhibition model by flow test resultsCulturing after 60 h,the ratio of Th17 cells of model control group,MTX control group,nitidine chloride in high,medium and low concentration groups were 1.7±0.141,0.367±0.058,0.5±0.082,0.875±0.126,1.325±0.096 respectively.The ratio of Th17 cells in the high,middle and low concentration groups were significantly reduced,compared with the control group(p<0.01).Nitidine chloride in medium concentration and low concentration group compared with MTX in the control group were statistically difference(p<0.01),and nitidine chloride in high concentration group compared with MTX control group had no statistical difference(p=0.127).Conclusion:1.The activated collagen and the inactivated collagen cannot stimulate the proliferation of the spleen Th17 cells in normal mice,but both can stimulate the proliferation of spleen Th17 cells in the CIA mice.IL-23 is a necessary cytokines for the proliferation of Th17 cells,but the addition of different levels of IL-23 has no effects on the proliferation of Th17 cells.2.Nitidine chloride has inhibitory effect on the proliferation of Th17 cells,and the ability to suppress Th17 cells proliferation increased with the increase of drug concentration.The high concentration of nitidine chloride was similar to that of the MTX control group on Th17 proliferation. |
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