摘要
目的 基于转录组数据分析发现与验证六味地黄方活性组分的抗补体作用,并研究其可能的作用途径。方法 人恶性黑素瘤细胞A375用DMEM/F12高糖培养基培养,分为细胞对照、六味地黄苷糖LW-AFC、寡糖组分CA-30、多糖组分LWB-B和糖苷组分LWD-b组(终浓度均为100 mg·L^(-1)),培养6 h后用L1000技术采集细胞转录组数据,采用Cosine相似度算法和特征方向法对各组转录组数据进行相似性比对和差异表达分析,采用基因富集分析算法进行KEGG通路富集分析。采用实时荧光定量PCR(RT-qPCR)测定补体通路基因C3和C5 m RNA表达。采用基于补体经典途径的溶血活性实验测定相对溶血率,观察LWAFC,CA-30,LWB-B和LWD-b(终浓度均为1,10和100 mg·L^(-1))及LWD-b中各主要成分莫诺苷、芍药苷、獐牙菜苷、马钱苷、马钱苷酸和丹皮酚原苷(终浓度均为1和100μmol·L^(-1))的抗补体活性。采用补体成分(C1q,C2,C3,C4,C5和C9)缺失血清法测定LWD-b的补体作用靶点。结果 转录组数据分析结果显示,LWD-b下调的基因显著富集于补体级联激活通路(P<0.01),而LW-AFC,CA-30和LWB-B此作用不明显。RT-qPCR结果表明,与细胞对照组相比,LWD-b 100 mg·L^(-1)组补体C3和C5 mRNA表达明显降低(P<0.01)。抗补体活性测定结果表明,与补体组相比,LWD-b各浓度组及LW-AFC 10和100 mg·L^(-1)组相对溶血率均明显降低(P<0.01),但仅LWD-b组相对溶血抑制率>20%,提示LWD-b具有抗补体活性,LW-AFC,CA-30和LWB-B此作用不明显。LWD-b中主要成分的抗补体活性筛选结果表明,与补体组相比,莫诺苷1和100μmol·L^(-1)组及芍药苷100μmol·L^(-1)组相对溶血率均明显降低(P<0.01),且相对溶血抑制率均>20%;獐牙菜苷、马钱苷、马钱苷酸和丹皮酚原苷组相对溶血率虽也明显降低(P<0.01),但其相对溶血抑制率均<20%;提示莫诺苷和芍药苷具有显著抗补体活性,而其余成分抗补体活性较弱。补体成分缺失血清法测定结果表明,分别与补体C1q,C2,C3,C5和C9缺失血清组相比,LWD-b 100 mg·L^(-1)处理后相对溶血率变化均<5%,提示LWD-b降低溶血能力与其作用于上述补体成分相关。结论 基于转录组数据分析发现并验证了六味地黄方活性组分LWD-b具有抗补体激活作用,莫诺苷和芍药苷可能是其发挥该作用的主要活性成分。LWD-b可能通过作用于补体成分C1q,C2,C3,C5和C9发挥作用。
OBJECTIVE To discover and verify the anti-complement effect of active components of Liuwei Dihuang formula based on transcriptome data analysis,and investigate the possible mechanisms.METHODS A375 cells were cultured in DMEM/F12 high glucose medium and divided into cell control,Liuwei Dihuang-active fraction combination LW-AFC,oligosaccharide component CA-30,polysaccharide component LWB-B and glycoside component LWD-b(the final concentration was 100 mg·L-1)groups.After incubation of 6 h,the transcriptome data was collected by L1000 technique before the cosine similarity algorithm was used to compare the similarity and characteristic direction approach was adopted to analyze the differential expression of transcriptome data in each group,and the gene enrichment analysis(GSEA)algorithm was employed for KEGG pathway enrichment analysis.The real time-quantitative PCR(RT-qPCR)was used to detect the expressions of complement genes C3 and C5 mRNA.The classical complement pathway based-hemolysis test was conducted to evaluate the anti-complement activities of LW-AFC,CA-30,LWB-B and LWD-b(the final concentrations were 1,10 and 100 mg∙L-1),as well as the main components of LWD-b,morroniside,paeoniflorin,sweroside,loganin,loganic acid and paeonolide(the final concentrations were 1,100μmol·L-1).The hemolytic targets of LWD-b were evaluated by individual complement(C1q,C2,C3,C4,C5 and C9)depleted sera.RESULTS Transcriptome data analysis showed that the genes down-regulated by LWD-b were significantly enriched in the complement cascade activation pathway(P<0.01),rather than in LW-AFC,CA-30 and LWB-B.RT-qPCR results showed that compared with the cell control group,the expressions of C3 and C5 mRNA in the LWD-b 100 mg∙L-1 group decreased significantly(P<0.01).The results of anti-complement activity test showed that compared with the complement group,the relative hemolysis rates of LWD-b(1,10 and 100 mg·L-1)and LW-AFC(10 and 100 mg·L-1)groups decreased(P<0.01),and the relative hemolysis inhibition rate was>20%only in the LWD-b group,but not in LWB-B and CA-30 groups,suggesting that only LWD-b had anti-complement activity.The screening results of anti-complement activities of components in LWD-b showed that compared with the complement group,the relative hemolysis rates in the groups of 1 and 100μmol·L-1 of morroniside and 100μmol·L-1 of paeoniflorin decreased(P<0.01),and the relative hemolysis inhibition rates were all>20%,while the relative hemolysis rates of sweroside,loganin,loganin acid and paeonolide decreased(P<0.01),but the relative hemolysis inhibition rates were all<20%,suggesting that morroniside and paeoniflorin had anti-complement activities,while the other components in LWD-b did not.The results of complement components depleted sera assay showed that the relative hemolysis rates were all<5%in LWD-b 100 mg·L-1 treatment groups,compared with the complements C1q,C2,C3,C5 and C9 depleted sera groups respectively,suggesting that the effect of LWD-b on reducing hemolysis depended on those complement components in sera.CONCLUSION The anti-complement activation effect of LWD-b is verified based on transcriptome data analysis.Morroniside and paeoniflorin may be the main components that contribute to the anti-complement effect that is possibly due to its interactions with complements C1q,C2,C3,C5 and C9.
作者
林颖
韩露
高圣乔
罗丹
肖智勇
周文霞
LIN Ying;HAN Lu;GAO Sheng-qiao;LUO Dan;XIAO Zhi-yong;ZHOU Wen-xia(Nanjing University of Chinese Medicine,Nanjing 210023,China;State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2023年第3期168-177,共10页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(82141218)
天津市科技发展计划项目(22ZYJDSS00080)。
关键词
六味地黄方
活性组分
LWD-b
补体系统
溶血实验
转录组数据分析
Liuwei Dihuang formula
active components
LWD-b
complement system
hemolysis test
transcriptome data analysis
作者简介
林颖,硕士研究生,主要从事药理学研究,E-mail:LY122483@163.com;通讯作者:肖智勇,E-mail:zy-xiao@163.com;通讯作者:周文霞,E-mail:zhouwx@nic.bmi.ac.cn。
