紫珠提取物通过抑制HMGB1水平改善小鼠宫颈上皮细胞炎症反应的机制

Mechanism of Callicarpa nudiflora extract improving the inflammatory response of mice cervical epithelial cells by inhibiting the level of HMGB1

目的探讨紫珠提取物通过抑制高迁移率族蛋白框1(HMGB1)水平改善小鼠宫颈上皮细胞炎症反应的机制。方法30只CD-1远交的孕鼠用于实验研究,通过脂多糖诱导早产小鼠模型。将成功怀孕的小鼠按照随机数字表法分成3组:对照组(正常孕鼠,等量0.9%氯化钠溶液灌胃,n=10),炎症模型组(早产模型,等量0.9%氯化钠溶液灌胃,n=10)和紫珠提取物组(早产模型,100 mg·kg^(-1)·d^(-1)紫珠提取物灌胃,n=10),共干预7 d。通过蛋白印迹分析小鼠宫颈组织HMGB1/TLR4/NF-κB的蛋白表达。通过酶联免疫吸附试验检测小鼠宫颈阴道粘液中细胞因子[肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-6、IL-8]的水平。通过RT-PCR分析小鼠宫颈组织焦亡相关蛋白iNOS、NLRP3和caspase-4的mRNA表达。通过水溶性荧光探针和可溶性E-钙粘蛋白(SECAD)水平评估上皮细胞通透性。结果炎症模型组HMGB1、TLR4和NF-κB的蛋白表达较对照组升高,紫珠提取物组HMGB1、TLR4和NF-κB的蛋白表达较炎症模型组降低,差异均有统计学意义(P<0.05)。炎症模型组TNF-α、IL-1β、IL-6和IL-8的水平较对照组升高,紫珠提取物组TNF-α、IL-1β、IL-6和IL-8的水平较炎症模型组降低,差异均有统计学意义(P<0.05)。炎症模型组iNOS、NLRP3和caspase-4的mRNA表达较对照组升高,紫珠提取物组iNOS、NLRP3和caspase-4的mRNA表达较炎症模型组降低,差异均有统计学意义(P<0.05)。炎症模型组SECAD水平和荧光强度较对照组升高,紫珠提取物组SECAD水平和荧光强度较炎症模型组降低,差异均有统计学意义(P<0.05)。结论脂多糖可以激活小鼠宫颈上皮细胞的炎症反应,这种炎症反应会导致宫颈上皮屏障受损,紫珠提取物通过抑制HMGB1表达降低小鼠宫颈上皮炎症反应,改善宫颈上皮屏障受损。

Objective To explore the mechanism of Callicarpa nudiflora extract improving the inflammatory response of mice cervical epithelial cells by inhibiting the level of high mobility group box 1(HMGB1).Methods Thirty CD-1 outbred pregnant mice were used for experimental research.The preterm mice models were induced by lipopolysaccharide.The successfully pregnant mice were divided into three groups according to the random number table method:control group(normal pregnant mice,gavage with equal volume of normal saline,n=10),inflammation model group(preterm labor model,gavage with equal volume of normal saline,n=10)and Callicarpa nudiflora extract group(preterm labor model,100 mg·kg^(-1)·d^(-1)Callicarpa nudiflora extract gavage,n=10),a total of 7 days of intervention.The expressions of HMGB1/TLR4/NF-κB protein in mice cervical tissue were analyzed by Western blotting.Cytokine[tumor necrosis factor alpha(TNF-α),interleukin(IL)-1β,IL-6,IL-8]levels in mice cervicovaginal mucus were detected by enzyme-linked immunosorbent assay.The mRNA expressions of pyroptosis-related proteins iNOS,NLRP3 and caspase-4 in mice cervical tissue were analyzed by RT-PCR.Epithelial cell permeability was assessed by water-soluble fluorescent probes and soluble E-cadherin(SECAD)levels.Results The expressions of HMGB1,TLR4,and NF-κB protein in the inflammation model group were higher than those in the control group,and the expressions of HMGB1,TLR4,and NF-κB protein in the Callicarpa nudiflora extract group were lower than those in the inflammation model group,the differences were statistically significant(P<0.05).The levels of TNF-α,IL-1β,IL-6 and IL-8 in the inflammation model group were higher than those in the control group,the levels of TNF-α,IL-1β,IL-6 and IL-8 in the Callicarpa nudiflora extract group were lower than those in the inflammation model group,the differences were statistically significant(P<0.05).The expressions of iNOS,NLRP3 and caspase-4 mRNA in the inflammation model group were higher than those in the control group,and the expressions of iNOS,NLRP3 and caspase-4 mRNA in the Callicarpa nudiflora extract group were lower than those in the inflammation model group,the differences were statistically significant(P<0.05).The level of SECAD and fluorescence intensity in the inflammation model group were higher than those in the control group,and the level of SECAD and fluorescence intensity in the Callicarpa nudiflora extract group were lower than those in the inflammation model group,and the differences were statistically significant(P<0.05).Conclusion Lipopolysaccharide can activate the inflammatory response of mice cervical epithelial cells,which can lead to the damage of the cervical epithelial barrier.The Callicarpa nudiflora extract reduces the inflammatory response of mice cervical epithelial cells by inhibiting the expression of HMGB1,and improves the damaged cervical epithelial barrier.