山茱萸水提液对胶原诱导关节炎模型小鼠滑膜组织中IL-12家族调控作用的实验研究

目的观察山茱萸水提液对胶原诱导的关节炎(collagen induced arthritis,CIA)小鼠的干预作用,并通过检测山茱萸水提液对CIA小鼠滑膜组织中IL-12、IL-23、IL-27及IL-35表达的影响,探讨山茱萸水提液通过调控IL-12家族发挥对CIA小鼠干预作用的机制,揭示"酸入肝,酸补肝"中药性味理论指导下的补肝养血疗法治疗痹证的分子机制。方法SPF级DBA/J小鼠,雄性,8~10周龄,40只。采用随机数字表法随机分为4组:空白对照组、模型对照组、山茱萸组、西药对照组,每组10只。然后对模型对照组、山茱萸组、西药对照组小鼠以Ⅱ型胶原诱导CIA小鼠模型,采用关节炎指数评分法进行模型评价,评价成功的小鼠开始给予药物干预,空白对照组不作处理;模型对照组给予等量生理盐水经口灌胃;山茱萸组给予山茱萸24 g/(kg·d)]悬液经口灌胃,1次/d,连续2周;西药对照组给予布洛芬0.08 g/(kg·d)]悬液经口灌胃,1次/d,连续2周。末次给药后2 h,评价各组小鼠关节炎指数评分并记录;然后将小鼠过量麻醉处死,取左侧膝关节,剥离滑膜组织,冻存于-80℃冰箱中,用于ELISA法检测滑膜组织中IL-12、IL-23、IL-27及IL-35的含量;同时取右侧膝关节,去皮肤及肌肉,浸泡于4%多聚甲醛溶液中,固定24 h以上,EDTA螯合脱钙剂脱钙,制备石蜡切片,采用IHC法检测滑膜组织中IL-12、IL-23、IL-27及IL-35的表达水平。结果与空白对照组比较,模型对照组小鼠关节炎指数评分及病理评分显著升高(P<0.01),差异有统计学意义;与模型对照组比较,山茱萸组、西药对照组关节炎指数评分及病理评分显著降低(P<0.01),差异有统计学意义;山茱萸组与西药对照组间关节炎指数评分及病理评分差异无统计学意义。与空白对照组比较,模型对照组小鼠膝关节滑膜组织中IL-12、IL-23表达水平显著升高(P<0.01),IL-27、IL-35表达水平显著降低(P<0.01),差异有统计学意义;与模型对照组比较,山茱萸组、西药对照组小鼠膝关节滑膜组织中IL-12、IL-23表达水平显著降低(P<0.01),IL-27、IL-35表达水平显著升高(P<0.01),差异有统计学意义;山茱萸组与西药对照组间膝关节滑膜组织中IL-12、IL-23、IL-27及IL-35表达水平差异无统计学意义。结论山茱萸水提液对CIA小鼠干预作用明显,能够明显降低关节炎指数评分及病理评分。该作用可能是通过抑制CIA小鼠膝关节滑膜组织中IL-12、IL-23,促进IL-27、IL-35的表达而实现的。

Experimental Study on Regulation of Water Extract of Shanzhuyu(Cornus Officinalis) on IL-12 Family in Synovial Tissue of CIA Mice

Objective To observe the effect of the water extract of Shanzhuyu(Cornus officinalis)on the expressions of IL-12,IL-23,IL-27 and IL-35 in the synovial tissue of CIA mice and study the mechanism of the control of IL-12 family water extract of Shanzhuyu(Cornus officinalis)so as to propose the molecular mechanism of liver-nourishing and blood-nourishing therapy in the treatment of Bi syndrome under the theory of traditional Chinese medicine of sour drugs can affect liver and nourish liver.Methods Forty SPF DBA/J male mice,8~10 weeks old,were randomly divided into four groups:blank control group,model control group,Shanzhuyu(Cornus officinalis)group and western medicine control group with 10 rats in each group.Then,the model control group,Shanzhuyu(Cornus officinalis)group and western medicine control group were induced by typeⅡcollagen,and the model was evaluated by arthritis index scoring method.The successful mice began to give drug intervention.The blank control group was not treated.The model control group was given the same amount of normal saline intraoral,and the Shanzhuyu(Cornus officinalis)24 g/(kg·d)]suspension was given intragastrically once a day for 2 weeks.In the western medicine control group,ibuprofen0.08 g/(kg·d)]suspension was given intragastrically once a day for 2 weeks.Two hours after the last administration,the arthritis index of mice in each group was evaluated and recorded,and then the mice were anesthetized and killed.The left knee joint was taken out,the synovial tissue was stripped and frozen in a refrigerator at-80℃,and the contents of IL-12,IL-23,IL-27 and IL-35 in synovial tissue were detected by ELISA method.At the same time,the right knee joint,skin and muscle were immersed in 4%paraformaldehyde solution,fixed for more than 24 hours,decalcified by EDTA chelating decalcification agent.Paraffin sections were prepared,and the expressions of IL-12,IL-23,IL-27 and IL-35 in synovial tissue were detected by IHC method.Results Compared with those of the blank control group,the arthritis index score and pathological score of the model control group were significantly higher(P<0.01),and the arthritis index score and pathological score of the Shanzhuyu(Cornus officinalis)group and the western medicine control group were significantly lower than those of the model control group(P<0.01),but there was no significant difference in arthritis index score or pathological score between the Shanzhuyu(Cornus officinalis)group and the western medicine control group.Compared with those of the blank control group,the expressions of IL-12 and IL-23 in synovial tissue of knee joint in model control group was significantly higher(P<0.01),and the expressions of IL-27 and IL-35 in synovial tissue of knee joint in model control group were significantly lower than that in blank control group(P<0.01).Compared with those of the model control group,the expressions of IL-12,IL-23,IL-27 and IL-35 in synovial tissue of knee joint in the Shanzhuyu(Cornus officinalis)group and western medicine control group were significantly lower(P<0.01),but there was no significant difference in the expressions of IL-12,IL-23,IL-27 or IL-35 in synovial tissue between the Shanzhuyu(Cornus officinalis)group and western medicine control group.Conclusion The water extract of Shanzhuyu(Cornus officinalis)has obvious intervention effect on CIA mice and can significantly reduce the arthritis index score and pathological score.This effect may be achieved by inhibiting IL-12 and IL-23 in synovial tissue of knee joint of CIA mice to promote the expressions of IL-27 and IL-35.