云南白药在P.g-LPS诱导的炎症环境下对人牙髓细胞分泌TNF-α、IL-6、IL-1β的影响

摘要

目的 研究云南白药在P.g-LPS诱导的炎症环境下对人牙髓细胞分泌IL-6、IL-lβ、TNF-α的影响,探索云南白药对牙髓炎抑制的影响.方法 采用改良组织块法体外培养人牙髓细胞,免疫组化鉴定细胞来源,10μg/mL的P.g-LPS刺激人牙髓细胞24 h后,分3组,空白对照组1%FBS培养液,阳性对照组10μg/mL P.g-LPS,白药组10μg/mL云南白药.于刺激结束,换液后1 d、3 d时ELISA检测培养上清中IL-6、IL-lβ、TNF-α水平,同时换液3 d时定量real-time PCR检测TNF-α、IL-6、IL-lβmRNA的表达.结果 成功培养HDPCs,波形丝蛋白表达阳性,角蛋白为阴性.P.g-LPS刺激人牙髓细胞1 d时,3组之间TNF-α、IL-6、IL-1β蛋白表达无差别,换液后1 d和3 d,空白对照组和白药组低于阳性对照组(P<0.05),白药组与空白对照组差异无统计学意义(P>0.05).然而换液后3 d白药组TNF-α、IL-6、IL-1βmRNA的表达低于空白对照组(P<0.05).结论 在P.g-LPS诱导的炎症环境下,云南白药作用3 d时,对HDPCs的TNF-α、IL-6、IL-1β蛋白分泌没有明显抑制作用,但抑制TNF-α、IL-6、IL-1βmRNA的表达.%Objective To investigate the effect of Yunnan Baiyao on the secretion of TNF-α,IL-6, IL-1βfrom human dental pulp cells(hDPCs)under lipopolysaccharide from Porphyromonas gingivalis (LPS-PG)induced inflammatory environment,and investigate the inhibition effect of Yunnan Baiyao on pulpitis. Methods HDPCs were cultured by the modified tissue explant culture technique. Cultured hDPCs were identified and verified by the immunocytochemical staining. HDPCs were exposed to 10μg/mL LPS-PG solution for 1 day, then the LPS-PG containing culture media were removed and collected,the hDPCs were divided into 3 groups cultured with different new culture media,the control group was cultured with 1%FBS MEM,the LPS-PG group was cultured with 10μg/mL LPS-PG,the Baiyao group was cultured with 10μg/mL Yunnan Baiyao. Culturing media were collected on exposure finishing,1st and 3rd day after the medium changing for detection of IL-6, IL-1β and TNF-αproteins by ELISA,and on 3rd day the quantitative Real-Time PCR(qRT-PCR)was performed for IL-6,IL-1β and TNF-αgene expression of the hDPCs.Results HDPCs culturing showed a positive vimentin expression and a negative keratin expression. 3 groups showed no difference in TNF-α,IL-6 and IL-1βsecretions immediately after the LPS-PG exposure for 1 day by ELISA,and the LPS-PG group showed higher secretions of TNF-α,IL-6 and IL-1βthan other groups on1st and 3rd day(P<0.05). Baiyao group and control group showed no differences(P>0.05). However,qRT-PCR showed lower gene expressions of TNF-α,IL-6,IL-1βin the Baiyao group than other groups at 3rd day(P<0.05).Conclusion Under inflammatory environment induced by LPS-PG,the Yunnan Baiyao shows no inhibitory effect on secretion of TNF-α,IL-6 and IL-1βon3rd day after the medium changing,but can downregulate the mRNA expression.