摘要
目的探讨易黄汤对脾虚湿热型宫颈癌小鼠皮下移植瘤环磷酸鸟苷-腺苷酸合成酶(cGAS)/干扰素刺激基因(STING)/干扰素调节因子-3(IRF-3)信号通路及相关免疫因子的影响。方法选取6~8周龄SPF级雌性BALB/c小鼠50只,随机分为造模组40只和空白组10只。空白组正常喂养,造模组通过湿热环境及高脂高糖饮食建立脾虚湿热型小鼠模型,当小鼠出现嗜卧懒动、易激惹、形体消瘦、进食及饮水量减少、毛发疏松粗糙、粪便时干时溏、肛温稍有增加则认为造模成功。将2×106个/mL对数生长期HPV16阳性人宫颈癌CaSki细胞悬液0.2 mL接种于上述50只小鼠右腋下,5~7 d后可触及直径≥5 mm结节时即认为皮下移植瘤造模成功。将造模组按随机数字表法分为模型组和易黄汤低剂量、中剂量、高剂量组,每组10只。低、中、高剂量组均按体质量40.0 mL/kg分别予含生药浓度0.25、0.50、1.00 g/mL易黄汤药液灌胃,模型组和空白组予同等剂量生理盐水灌胃,均隔日1次,干预4周。干预后计算5组小鼠胸腺、脾脏指数和瘤质量、抑瘤率;ELISA法检测5组小鼠血清白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;支链DNA信号扩增法检测5组小鼠皮下移植瘤组织HPV E6 mRNA转录水平;Western blot测定5组小鼠移植瘤组织中cGAS、STING、IRF-3蛋白表达量。结果与空白组比较,模型组胸腺和脾脏指数、IL-6、TNF-α水平显著下降(P<0.05),HPV E6 mRNA转录水平显著增加(P<0.05);易黄汤不同剂量组中以高剂量组的抑瘤率最高为(35.42±4.86)%(P<0.05);与模型组比较,低、中、高剂量组胸腺和脾脏指数、IL-6、TNF-α水平及cGAS、STING和IRF-3蛋白表达量显著增加(P<0.05),皮下移植瘤质量及HPV E6 mRNA转录水平显著降低(P<0.05),且降低程度与易黄汤剂量成正比。结论脾虚湿热能促进CaSki细胞生长、HPV表达增加,易黄汤可能通过激活cGAS/STING/IRF-3信号通路增强免疫功能,从而抑制HPV表达及宫颈癌小鼠移植瘤生长。
Objective:To investigate the effect of Yihuang Decoction on cGAS/STING/IRF-3 signal pathway and related immune factors in cervical cancer mice with spleen deficiency and damp-heat syndrome.Methods:A total of 50 SPF female BALB/c mice aged 6-8 weeks were selected and randomly divided into spleen deficiency and damp-heat syndrome model group(SDDH group,n=40)and blank group(n=10).The mouse model of spleen deficiency and damp-heat syndrome was established by hot and humid environment and high-fat and high-sugar diet in the SDDH group.The mice showed lethargy,irritability,weight loss,reduced amount of food and drinking,loose and rough hair,and feces were sometimes dry and sometimes loose with a slight increase in anal temperature,which meant the modeling was considered successful,while the blank group was fed normally.0.2 mL of 2×106/mL logarithmic growth phase HPV16-positive human cervical cancer CaSki cell suspension was inoculated into the right axilla of 50 mice.The subcutaneous transplanted tumor model was successful when nodules with a diameter of 5 mm or greater could be palpated after 5-7 days.According to the random number table method,the SDDH group was randomly divided into a model group and low,medium,and high-dose groups of Yihuang Decoction,with 10 mice in each group.The Yihuang decoction was given to three different dose groups through intragastric administration with concentrations of crude drug of 0.25,0.50,and 1.00 g/mL based on body mass,at a rate of 40.0 mL/kg.The model and blank groups were given the same dose of saline solution.The time of intervention was once every other day for 4 weeks.After the intervention,the researchers calculated the indexes of the thymus and spleen,tumor mass and tumor inhibition rate.They also detected the level of serum interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)using the ELISA method.The HPV E6 mRNA transcription level of subcutaneously transplanted tumor tissue in the five groups was detected by branched chain DNA signal amplification.Additionally,the expression of cGAS,STING,and IRF-3 protein in transplanted tumor tissue of the five groups was determined by Western blot.Results:The model group showed a significant decrease in thymus and spleen index,as well as level of IL-6 and TNF-αcompared to the blank group.The transcription level of HPV E6 mRNA was found to be significantly increased in the model group,while the highest tumor inhibition rate was observed in the high dose group at(35.42±4.86%)(P<0.05);Compared with the model group,the low,middle,and high dose groups showed a significant increase in thymus and spleen index,level of IL-6,TNF-α,and the protein expression of cGAS,STING,and IRF-3.Additionally,the tumor mass and HPV E6 mRNA transcription level significantly decreased in the low,middle,and high dose groups,which was found to be proportional to the dose of Yihuang Decoction.(P<0.05).Conclusion:Spleen deficiency and dampness-heat can promote the growth of CaSki cells and increase the expression of HPV.Yihuang Decoction may enhance immune function by activating cGAS/STING/IRF-3 signaling pathway,thus inhibiting the expression of HPV and the growth of cervical cancer in mice.
作者
林兰
吴荣莉
郑怡真
吴冬梅
LIN Lan;WU Rongli;ZHENG Yizhen;WU Dongmei(The Second People'S Hospital Affiliated to Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350003,China)
出处
《福建中医药》
2024年第4期10-14,共5页
Fujian Journal of Traditional Chinese Medicine
基金
福建省自然科学基金面上项目(2022J01344)
国家中医药管理局第五批全国中医临床优秀人才研修项目(国中医药人教函[2022]1号)。
