黄芩苷调节PI3K/AKT通路对LPS诱导大鼠流产子宫巨噬细胞的影响

Effects of baicalin on uterine macrophages in LPS-induced abortion animal model by regulating PI3K/AKT pathway and its mechanism

目的:探究黄芩苷调节PI3K/AKT通路对脂多糖(lipopolysaccharid,LPS)诱导大鼠流产子宫巨噬细胞的影响。方法:将60只大鼠分为对照组、LPS组、LPS+黄芩苷组和LPS+黄芩苷+LY294002组(n=15)。尾静脉注射LPS诱导流产模型,再由尾静脉注射黄芩苷进行干预,腹腔注射LY294002(1 mg·kg^(-1))抑制PI3K/AKT通路。在妊娠第10天对大鼠进行安乐死观察胚胎发育情况,计算流产率和胚胎吸收率。通过ELISA和流式细胞术检测各组血清炎症细胞因子及子宫巨噬细胞水平。利用免疫组化检测巨噬细胞M1极化标志蛋白CD86和M2极化标志蛋白CD206的水平。通过RT-qPCR和Western blotting检测PI3K、AKT转录及翻译水平。结果:4组的各项指标比较差异均具有统计学意义(均P<0.05)。LPS+黄芩苷组的胚胎吸收6例,流产率5例,均显著低于LPS组(均为15例,P<0.05)。LPS组的TNF-α水平[(397.53±48.27)pg·ml^(-1)]、IL-1β水平[(61.28±8.34)pg·ml^(-1)]和子宫巨噬细胞比例显著高于对照组(均P<0.05)。LPS+黄芩苷组的TNF-α水平[(214.86±25.14)pg·ml^(-1)]、IL-1β水平[(49.74±6.79)pg·ml^(-1)]和子宫巨噬细胞比例显著低于LPS组(均P<0.05)。LPS组的M1型细胞比例显著高于对照组,而M2型细胞比例以及PI3K、AKT mRNA表达水平显著低于对照组(均P<0.05)。LPS+黄芩苷组的M1型细胞比例显著低于对照组,而M2型细胞比例以及PI3K、AKT mRNA表达水平显著高于对照组(均P<0.05)。LPS+黄芩苷+LY294002组的胚胎吸收率(93.33%)和流产率(86.67%)均显著高于LPS+黄芩苷组(均P<0.05)。LPS+黄芩苷+LY294002组的TNF-α水平[(362.75±40.29)pg·ml^(-1)]、IL-1β水平[(57.62±7.67)pg·ml^(-1)]、巨噬细胞比例、M1型细胞比例显著高于LPS+黄芩苷组,M2型细胞比例以及PI3K、AKT蛋白水平显著低于LPS+黄芩苷组(均P<0.05)。结论:黄芩苷可能通过调节PI3K/AKT通路来降低子宫巨噬细胞比例并诱导其向M2型极化和抑制向M1型极化,从而抑制流产。

Objective:To explore the effect of baicalin's regulation of PI3K/AKT pathway on lipopolysaccharid(LPS)induced abortion uterine macrophages in rats.Methods:60 rats were divided into control group,LPS group,LPS+baicalin group,and LPS+baicalin+LY294002 group(n=15).A miscarriage model was induced by LPS injection through the tail vein.Intervention was performed by injecting baicalin through the tail vein.The PI3K/AKT pathway was inhibited by intraperitoneal injection of LY294002(1 mg·kg^(-1)).On the 10th day of pregnancy,the rats were euthanized to observe the embryonic development.The abortion rate and embryo absorption rate were calculated.The levels of serum inflammatory cytokines and uterine macrophages were detected by ELISA and flow cytometry.Immunohistochemical staining was used to detect the level of macrophage M1 polarization marker protein CD86 and M2 polarization marker protein CD206,so as to analyze the polarization of macrophages in decidua tissue.The transcription and translation levels of PI3K and AKT were detected by RT-qPCR and Western blotting.Results:The differences in the indicators of the four groups were statistically significant(all P<0.05).The embryo absorption of the LPS+baicalin group were 6 cases,and the abortion were 5 cases,which were significantly lower than those of the LPS group(15 cases,all P<0.05).The proportion of TNF-α[(397.53±48.27)pg·ml^(-1)],IL-1β[(61.28±8.34)pg·ml^(-1)]and uterine macrophages in the LPS group were significantly higher than those in the control group(all P<0.05).The proportion of TNF-α[(214.86±25.14)pg·ml^(-1)],IL-1β[(49.74±6.79)pg·ml^(-1)]and uterine macrophages in the LPS+baicalin group were significantly lower than those in the LPS group(all P<0.05).The proportion of M1 cells in the LPS group was significantly higher than that in the control group,while the proportion of M2 cells and the expression levels of PI3K and AKT mRNA were significantly lower than those in the control group(all P<0.05).The proportion of M1 cells in the LPS+baicalin group was significantly lower than that in the control group,while the proportion of M2 cells and the expression levels of PI3K and AKT mRNA were significantly higher than those in the control group(all P<0.05).The embryo absorption(14 cases)and abortion(13 cases)of the LPS+baicalin+LY294002 group were significantly higher than those of the LPS+baicalin group(all P<0.05).The proportion of TNF-α[(362.75±40.29)pg·ml^(-1)],IL-1β[(57.62±7.67)pg·ml^(-1)],macrophages,the proportion of M1 type cells in the LPS+baicalin+LY294002 group were significantly higher than those in the LPS+baicalin group,M2 type The cell ratio,PI3K and AKT protein levels were significantly lower than those in the LPS+baicalin group(P<0.05).Conclusion:Baicalin may promote the PI3K/AKT pathway to reduce the proportion of uterine macrophages and induce uterine macrophages to polarize to M2 and inhibit M1 thus to inhibit abortion.