壮骨健膝方含药血清对脂多糖诱导兔膝滑膜巨噬细胞炎症反应的效应浓度研究

Effect of Concentration of Serum Containing Zhuanggu Jianxi Decoction on Inflammatory Response of Rabbit Knee Synovial Macrophage Induced by Lipopolysaccharide

目的探讨壮骨健膝方含药血清对脂多糖(LPS)诱导兔膝滑膜巨噬细胞炎症反应的效应浓度。方法将12只新西兰大白兔随机分为壮骨健膝方组6只和空白组6只,壮骨健膝方组予壮骨健膝方药液灌胃,空白组予生理盐水灌胃,持续3 d。于末次给药后1 h进行腹腔注射麻醉,行腹主动脉采血,离心后获得壮骨健膝方兔含药血清和空白血清。分别制备5%、10%、15%、20%、25%壮骨健膝方兔含药血清完全培养液(含药完培)与兔空白血清完全培养液(空白完培)备用。(1)筛选含药血清干预浓度实验:体外培养兔膝滑膜巨噬细胞,随机分为对照组、空白血清组及含药血清组,分别采用普通完全培养基、空白完培(5%、10%、15%、20%、25%)和含药完培(5%、10%、15%、20%、25%)培养24 h,采用CCK8法检测各组细胞活性。(2)筛选LPS诱导致炎时间实验:将兔膝滑膜巨噬细胞随机分为空白组和LPS组,空白组加入普通完培,LPS组加入含1.0μg/mL的LPS培养液,分别培养12、24、36、48 h后,收集各组细胞上清液,ELISA法检测IL-1β、TNF-α含量。(3)采用含1μg/mL的LPS培养液刺激兔膝滑膜巨噬细胞12 h诱导其炎症反应,诱导成功后随机分为模型组和5%、10%、15%含药血清组,另取正常兔膝滑膜巨噬细胞作为空白组。模型组采用10%兔空白完培干预,5%、10%、15%含药血清组分别采用5%、10%、15%壮骨健膝方兔含药完培干预,空白组采用10%兔空白完培干预,均干预24 h。ELISA法检测各组细胞上清中白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶-3(MMP-3)、MMP-13的含量。结果(1)筛选含药血清干预浓度实验结果显示,5%、10%、15%含药血清组细胞活性与同浓度下空白血清组比较,差异无统计学意义(P>0.05)。(2)筛选LPS诱导致炎时间实验结果显示,LPS组内各时间点比较,刺激12 h后滑膜巨噬细胞上清液中IL-1β、TNF-α的含量最高(P<0.05)。(3)与空白组比较,模型组IL-1β、TNF-α、MMP-3、MMP-13含量明显升高(P<0.05);与模型组比较,10%、15%含药血清组IL-1β、TNF-α、MMP-3、MMP-13含量均明显降低(P<0.05)。结论10%、15%壮骨健膝方兔含药血清为干预LPS诱导兔膝滑膜巨噬细胞炎症反应的效应浓度,以10%浓度更为经济适宜。

Objective: To explore the effect concentration of serum containing Zhuanggu Jianxi Decoction on the inflammatory response of rabbit knee synovial macrophages induced by lipopolysaccharide(LPS). Methods: Twelve New Zealand white rabbits were randomly divided into Zhuanggu Jianxi Decoction group(n=6) and blank group(n=6). Zhuanggu Jianxi Decoction group was given Zhuanggu Jianxi Decoction solution by gavage, and the blank group was given normal saline by gavage for 3 days. One hour after the last administration, intraperitoneal injection anesthesia was performed, abdominal aorta blood was collected, and drug containing serum of Zhuanggu Jianxi Decoction rabbits and blank serum were obtained after centrifugation.The 5%, 10%, 15%, 20%, 25% Zhuanggu Jianxi Decoction rabbit drug-containing serum complete culture medium and rabbit blank serum complete culture medium were prepared.(1) The screening experiment of intervention concentration of drug containing serum was carried out. Rabbit knee synovial macrophages were cultured in vitro and randomly divided into control group, blank serum group and drug-containing serum group. They were cultured in normal complete culture medium, blank serum complete culture medium(5%, 10%, 15%, 20%, 25%) and drug-containing serum complete culture medium(5%, 10%, 15%, 20%, 25%) for 24 hours respectively. The cell activity of each group was detected by CCK8 method.(2) The time screening experiment of LPS induced inflammation was carried out. The rabbit knee synovial macrophages were randomly divided into blank group and LPS group. The blank group was added with normal complete culture medium, and the LPS group was added with LPS medium containing 1.0 μg/mL for 12, 24, 36 and 48 h respectively. The supernatant of each group was collected, and the contents of IL-1βand TNF-α were detected by ELISA.(3) The rabbit knee synovial macrophages were stimulated with 1.0 μg/mL LPS culture medium for 12 hours to induce inflammatory response. After successful induction, they were randomly divided into model group and 5%, 10% and 15% drug containing serum group, and normal rabbit knee synovial macrophages were taken as the blank group. The model group was intervened with 10% blank rabbit serum complete culture medium, and the 5%, 10% and 15% drug containing serum group were intervened with 5%, 10% and 15% Zhuanggu Jianxi Decoction rabbit drug containing serum complete culture medium respectively, and the blank group was intervened with 10% blank rabbit serum complete culture medium. After 24 hours, the contents of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), matrix metalloproteinase-3(MMP-3)and MMP-13 in cell supernatant of each group were detected by ELISA. Results:(1) The results of screening the intervention concentration of drugcontaining serum showed that there was no significant difference in cell activity between 5%, 10% and 15% drug-containing serum groups and blank serum group at the same concentration(P>0.05).(2) The results of LPS-induced inflammatory time screening showed that the contents of IL-1β and TNF-α in supernatant of synovial macrophages after 12 hours stimulation were the highest(P<0.05).(3) Compared with blank group, the contents of IL-1β, TNF-α, MMP-3 and MMP-13 in model group were significantly increased(P<0.05);Compared with model group, the contents of IL-1β, TNF-α,MMP-3 and MMP-13 in 10% and 15% drug-containing serum groups were significantly decreased(P<0.05). Conclusion: 10% and 15% Zhuanggu Jianxi Decoction rabbit drug containing serum were the effective concentration of LPS induced inflammatory response of rabbit synovial macrophages, and 10% was more economical.