摘要
目的基于“胰脾同源”理论探讨大建中汤通过IL-6/STAT3通路抑制髓源抑制性细胞(MDSCs)治疗胰腺癌的作用机制。方法将74只C57BL/6J小鼠随机分为模型对照组、吉西他滨组(40 mg·kg^(-1))及大建中汤低、中、高剂量组(1.84、3.69、7.38 g·kg^(-1)·d^(-1)),每组10只;同型对照组、大建中汤同型对照组、MDSCs耗竭组、大建中汤MDSCs耗竭组,每组6只。采用原位注射对数生长期Panc02-luc细胞(5×10^(5)个/只)复制小鼠胰腺癌原位模型。造模后次日开始,吉西他滨组腹腔注射给药,每次40 mg·kg^(-1),1周2次;大建中汤各剂量组按照上述剂量灌胃给药,每日1次,持续给药4周。同型对照组给予IgG抗体腹腔注射(10 g·kg^(-1)),隔日注射,注射体积为0.2 mL;大建中汤同型对照组在同型对照组干预的基础上,给予中剂量大建中汤(3.69 g·kg^(-1))灌胃,每日1次;MDSCs耗竭组给予Gr-1抗体腹腔注射(10 g·kg^(-1)),隔日注射,注射体积为0.2 mL;大建中汤MDSCs耗竭组在MDSCs耗竭组干预的基础上,给予中剂量大建中汤(3.69 g·kg^(-1))灌胃,每日1次;各组均持续给药2周。检测并记录小鼠生存期与体质量;采用小鼠活体成像检测肿瘤生长情况;检测大建中汤含药培养基pH值、渗透压值;采用MTT法测定细胞活力;流式细胞术检测胰腺肿瘤及脾脏组织中MDSCs、CD3^(+)T细胞、树突状细胞(DCs)、巨噬细胞及自然杀伤细胞(NKs)的细胞占比;RT-qPCR法检测肿瘤MDSCs免疫抑制功能相关基因的表达水平;网络药理学分析大建中汤调控MDSCs治疗胰腺癌的核心靶点;从GEO数据库下载GSE217845数据集,进行人类胰腺癌单细胞分析;Western Blot法检测肿瘤MDSCs中STAT3、Arg-1、S100A9蛋白表达水平。结果(1)与模型对照组比较,大建中汤低、中剂量组及吉西他滨组原位胰腺癌小鼠的生存期明显延长(P<0.05,P<0.01);大建中汤中剂量组小鼠在第18、25天的胰腺肿瘤组织荧光强度及肿瘤质量均显著降低(P<0.01)。(2)与空白组(DMEM完全培养基)比较,不同浓度大建中汤对胰腺癌细胞培养基pH值、渗透压及PANC-1、Panc02胰腺癌细胞活性均无明显的影响(P>0.05)。(3)与模型对照组比较,大建中汤中剂量组小鼠脾脏及肿瘤组织中的MDSCs数量显著降低(P<0.01),CD3^(+)T细胞显著增加(P<0.05,P<0.01),巨噬细胞及NKs细胞数量无明显变化(P>0.05),肿瘤组织中的DCs细胞数量显著增加(P<0.01)。(4)与同型对照组比较,在第17天大建中汤同型对照组小鼠的胰腺肿瘤荧光强度明显降低(P<0.05),MDSCs细胞数量显著下降(P<0.01),CD3^(+)T细胞数量显著增加(P<0.01)。与MDSCs耗竭组比较,在第3、17天大建中汤MDSCs耗竭组小鼠的胰腺肿瘤荧光强度无明显变化(P>0.05),MDSCs及CD3^(+)T细胞数量亦无明显变化(P>0.05)。(5)网络药理学分析显示,STAT3、IL-6是大建中汤调控MDSCs治疗胰腺癌的核心靶点;单细胞分析显示,MDSCs中富集了炎症、TNFα/NF-κB、IL-6/JAK/STAT3等信号通路,且MDSCs也是富集IL6/JAK/STAT3通路的主要细胞群体之一。(6)与模型对照组比较,大建中汤中剂量组胰腺癌小鼠肿瘤组织中MDSCs的Arg-1、S100A8、S100A9、IL-6、STAT3 mRNA表达及STAT3、Arg-1、S100A9蛋白表达均显著下调(P<0.05,P<0.01),NOS2 mRNA表达无明显变化(P>0.05)。结论大建中汤可有效延长胰腺癌小鼠生存期,延缓胰腺癌进展,其可能是通过抑制IL-6/STAT3通路,减少胰腺癌小鼠脾脏及肿瘤组织中的MDSCs浸润,下调其免疫抑制功能而实现的。
Objective To investigate the mechanism of Dajianzhong Decoction(DJZD)in treating pancreatic cancer by inhibiting myeloid-derived suppressor cells(MDSCs)through the IL-6/STAT3 pathway,based on the“Spleen-Pancreas Homology”theory.Methods A total of 74 C57BL/6J mice were randomly divided into the model control group,the Gemcitabine group(40 mg·kg^(-1)),and the low-,medium-,and high-dose DJZD Decoction groups(1.84,3.69,7.38 g·kg^(-1)·d^(-1),respectively),with 10 mice in each group;the isotype control group,the DJZD Decoction isotype control group,the MDSCs depletion group,and the DJZD Decoction MDSCs depletion group,with 6 mice in each group.Orthotopic pancreatic cancer models were established by injecting Panc02-luc cells(5×10^(5) cells/mouse)in the logarithmic growth phase into the pancreas.Starting from the day after modeling,the Gemcitabine group received intraperitoneal injections at 40 mg·kg^(-1)twice weekly;the DJZD Decoction groups received oral gavage at the aforementioned doses once daily,with all treatments continuing for 4 weeks.The isotype control group received intraperitoneal injections of IgG antibody(10 g·kg^(-1))every other day at an injection volume of 0.2 mL;the DJZD Decoction isotype control group received medium-dose DJZD Decoction(3.69 g·kg^(-1))by oral gavage once daily in addition to the isotype control intervention;the MDSCs depletion group received intraperitoneal injections of Gr-1 antibody(10 g·kg^(-1))every other day at an injection volume of 0.2 mL;the DJZD Decoction MDSCs depletion group received medium-dose DJZD Decoction(3.69 g·kg^(-1))by oral gavage once daily in addition to the MDSCs depletion intervention;all treatments continued for 2 weeks.Survival time and body mass were monitored and recorded;tumor growth was assessed using in vivo imaging;the pH and osmotic pressure of DJZD-containing medium were measured;cell viability was determined by MTT assay;flow cytometry was used to analyze the percentages of MDSCs,CD3^(+)T cells,dendritic cells(DCs),macrophages,and natural killer cells(NKs)in pancreatic tumors and spleen tissues;RT-qPCR was used to detect the expression levels of MDSCs immunosuppression-related genes in tumors;network pharmacology analysis was performed to identify the core targets of DJZD Decoction in regulating MDSCs for pancreatic cancer treatment;single-cell analysis of human pancreatic cancer was conducted using the GEO dataset GSE217845;Western Blot was used to detect the protein expression levels of STAT3,Arg-1,and S100A9 in tumor MDSCs.Results(1)Compared with the model control group,the low-and medium-dose DJZD Decoction groups and the Gemcitabine group showed significantly prolonged survival in orthotopic pancreatic cancer mice(P<0.05,P<0.01).The medium-dose DJZD Decoction group exhibited significantly reduced fluorescence intensity and tumor mass of pancreatic tumor tissues on days 18 and 25(P<0.01).(2)Compared with the blank group(DMEM complete medium),different concentrations of DJZD Decoction had no significant effects on the pH value and osmotic pressure of pancreatic cancer cell culture medium,nor on the viability of PANC-1 and Panc02 pancreatic cancer cells(P>0.05).(3)Compared with the model control group,the medium-dose DJZD Decoction group showed significantly decreased MDSCs numbers in spleen and tumor tissues(P<0.01),significantly increased CD3^(+)T cells(P<0.05,P<0.01),no significant changes in macrophages and NKs cells(P>0.05),and significantly increased DCs numbers in tumor tissues(P<0.01).(4)Compared with the isotype control group,the isotype control group treated with DJZD Decoction showed significantly reduced pancreatic tumor fluorescence intensity(P<0.05),significantly decreased MDSCs numbers(P<0.01),and significantly upregulated CD3^(+)T cells numbers on day 17(P<0.01).Compared with the MDSCs depletion group,the MDSCs depletion group treated with DJZD Decoction showed no significant changes in pancreatic tumor fluorescence intensity(P>0.05),MDSCs numbers or CD3^(+)T cells numbers on days 3 and 17(P>0.05).(5)Network pharmacological analysis revealed that STAT3 and IL-6 were core targets of DJZD Decoction in regulating MDSCs for pancreatic cancer treatment.Single-cell analysis showed that MDSCs were enriched with inflammatory pathways,TNFα/NF-κB and IL-6/JAK/STAT3 signaling pathways,and MDSCs were also one of the main cell populations enriched with the IL6/JAK/STAT3 pathway.(6)Compared with the model control group,the medium-dose DJZD Decoction group showed significantly downregulated mRNA expression of Arg-1,S100A8,S100A9,IL-6,and STAT3,as well as significantly decreased protein expression of STAT3,Arg-1,and S100A9 in tumor tissues of pancreatic cancer mice(P<0.05,P<0.01),while NOS2 mRNA expression showed no significant change(P>0.05).Conclusion DJZD prolongs survival time and delays pancreatic cancer progression by suppressing MDSCs infiltration and immunosuppression via the IL-6/STAT3 pathway.
作者
刘燕
王倩蕾
苏琳
吕春博
张飞
刘洺希
邹怡萌
胥孜杭
邹纯朴
LIU Yan;WANG Qianlei;SU Lin;LYU Chunbo;ZHANG Fei;LIU Mingxi;ZOU Yimeng;XU Zihang;ZOU Chunpu(School of Basic Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Longhua Hospital Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China)
出处
《中药新药与临床药理》
北大核心
2025年第7期1043-1055,共13页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
国家自然科学基金项目(82274229)
上海市2020年度“科技创新行动计划”生物医药科技支撑专项(20ZR1458500)
上海市青年科技启明星项目(23QA1409100)
中国博士后科学基金会面上资助项目(2023M732334)。
关键词
胰脾同源
大建中汤
胰腺癌
髓源抑制性细胞
IL-6/STAT3通路
免疫抑制
小鼠
spleen-pancreas homology
Dajianzhong Decoction
pancreatic cancer
myeloid-derived suppressor cells(MDSCs)
IL-6/STAT3 pathway
immunosuppression
mice
作者简介
刘燕,女,硕士研究生,研究方向:基于经典理论的中医药抗肿瘤研究。Email:lyfx99@icloud.com;王倩蕾,女,博士,副主任医师,研究方向:中医药防治消化道疾病相关研究。Email:qianleilh@126.com;通信作者:邹纯朴,男,教授,博士研究生导师,研究方向:内经理论与应用。Email:chunpuzou@shutcm.edu.cn;通信作者:胥孜杭,女,副教授,硕士研究生导师,研究方向:中医药调控肿瘤微环境的免疫机制研究。Email:xuzihang6207@shutcm.edu.cn。
