摘要
目的构建气虚湿瘀肾纤维化病证结合小鼠模型并对模型进行评价,运用多组学阐释其蛋白和代谢通路变化。方法24只C57BL/6J小鼠随机分为正常(N)组、模型(M)组、气虚湿瘀肾纤维化病证结合(BZ)组,每组8只,实验周期6周。采用“环孢素+高脂饮食+游泳力竭+恒温恒湿”建立气虚湿瘀肾纤维化小鼠模型,通过观察一般体征,测定肾功能、舌象RGB(红、绿、蓝)值、血液流变学指标、血脂、炎症和氧化指标,结合肾组织HE、Masson、PAS、油红O染色、TUNEL细胞凋亡和TGF-β免疫荧光对该模型进行评价;肾蛋白质组学联合血清代谢组学筛选出差异蛋白和代谢物并进行通路富集分析。结果BZ组小鼠第3周开始出现体质量下降(P<0.05),第4周体质量显著下降(P<0.01),同时出现进食及饮水量减少,毛发杂乱光泽度下降,精神萎靡,活动度下降,大便质稀。BZ组Scr、BUN、UACR、NAG均较N组升高(P<0.05或P<0.01),Scr、NAG水平与M组比较有统计学意义。BZ组小鼠舌象R值显著低于N组(P<0.01),B值高于N组,且均与M组有显著性差异(P<0.05)。BZ组小鼠全血多切变率黏度、HCT较N组和M组均升高,PV较N组升高(P<0.05或P<0.01)。BZ组TC、LDL-C、CRP、IL-6、MDA水平较N组和M组均显著升高(P<0.01),SOD活性较N组下降(P<0.05)。BZ组小鼠可见肾小管胞质空泡化明显,炎性细胞浸润、肾小球基底膜增厚、胶原纤维增生和脂质累积显著。BZ组肾组织细胞凋亡及TGF-β沉积增加。BZ组和N组共有299个差异蛋白,其中180个上调,119个下调;323个差异代谢物,其中205个上调,118个下调;初级胆汁酸生物合成、牛磺酸和亚牛磺酸代谢、不饱和脂肪酸的生物合成通路被差异蛋白和差异代谢物共同富集到,共涉及3个差异蛋白,9个差异代谢物,其中二十二碳五烯酸、二十碳五烯酸、牛磺酸、3-亚磺酰基丙氨酸、牛磺胆酸、酰基辅酶A氨基酸N-酰基转移酶1和酰基辅酶A氨基酸N-酰基转移酶2、超长链3-氧酰辅酶A还原酶表现出较高的预测准确性。结论通过“环孢素+高脂饮食+游泳力竭+恒温恒湿”法构建气虚湿瘀肾纤维化(RF)动物模型具有可行性。气虚湿瘀肾纤维化中不饱和脂肪酸生物合成与牛磺酸和亚牛磺酸代谢通路可能发挥重要作用。
Objective To construct and evaluate a mouse model of renal fibrosis(RF)combined with Qi deficiency and dampness stasis,and investigate the changes in protein and metabolic pathways using multiomics.Methods Twenty-four C57BL/6J mice were divided randomly into normal(N),model(M),and RF and syndrome combined groups(BZ)(n=8/group).The experiment lasted 6 weeks.A mouse model of RF with Qi deficiency and dampness stasis was established by“cyclosporine A+high-fat diet+swimming exhaustion+constant temperature and humidity”.The model was evaluated by detecting general signs,renal function,tongue RGB(red,green,blue)values,hemorheology indexes,blood lipids,and inflammation and oxidation indexes,combined with hematoxylin and eosin,Masson,periodic acid-Schiff,and Oil red O staining,terminal deoxynucleotidyl transferase dUTP nick end labeling apoptosis,and transforming growth factor-βimmunofluorescence analysis of renal tissue.Differential proteins and metabolites were screened by renal proteomics combined with serum metabolomics and subjected to pathway enrichment analysis.Results Body mass of mice in the BZ group began to decline at week 3(P<0.05)and decreased significantly at week 4(P<0.01),while food and water consumption decreased,the fur became messy and less glossy,mood and activity decreased,and stools became watery.Serum creatinine,blood urea nitrogen,urine albumin-creatinine ratio,and N-acetyl-beta-glucosaminidase(NAG)were significantly higher in the BZ group compared with those in the N group(P<0.05,P<0.01),and serum creatinine and NAG levels were significantly different compared with those in the M group.The R value of tongue images was significantly lower in the BZ group compared with that in the N group(P<0.01),while the B value was significantly higher(P<0.05).The viscosity of the whole blood multi-shear rate and the hematocrit were higher in the BZ group compared with those in the N and M groups,and the platelet volume was higher than in the N group(P<0.05,P<0.01).Total cholesterol,low-density lipoprotein cholesterol,C-reactive protein,interleukin-6,and malondialdehyde levels were significantly increased in the BZ group compared with those in the N and M groups(P<0.01),and superoxide dismutase activity was significantly decreased compared with that in the N group(P<0.05).Renal tubule vacuolation,inflammatory cell infiltration,glomerular basement membrane thickening,collagen fiber hyperplasia,and lipid accumulation were evident,and renal cell apoptosis and transforming growth factor-βdeposition were increased in the BZ group.There were 299 differential proteins in the BZ and N groups,including 180 up-regulated and 119 down-regulated proteins,and 323 differential metabolites,including 205 up-regulated and 118 down-regulated.Primary bile acid biosynthesis,taurine and hypotaurine metabolism,and biosynthesis of unsaturated fatty acids were co-enriched in differential proteins and differential metabolites,involving three differential proteins and nine differential metabolites.Among these,docosapentaenoic acid(22n-3),eicosapentaenoic acid,taurine,3-sulfinoalanine,taurocholic acid,Acnat1,Acnat2,and Hsd17b12 showed high prediction accuracy.Conclusions We successfully constructed an RF animal model of Qi deficiency and dampness stasis using the“cyclosporine A+high-fat diet+exhaustion of swimming+constant temperature and humidity”method.Biosynthesis of unsaturated fatty acids and taurine and hypotaurine metabolism may play important roles in this RF mouse model of Qi deficiency and dampness stasis.
作者
高冉冉
韩聪
连梦慧
李伟
GAO Ranran;HAN Cong;LIAN Menghui;LI Wei(First Clinical Medical School,Shandong University of Traditional Chinese Medicine,Ji’nan 250014;China.2.Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Ji’nan 250014;.3.Shandong University of Traditional Chinese Medicine,Ji’nan 250014)
出处
《中国比较医学杂志》
北大核心
2025年第4期43-57,共15页
Chinese Journal of Comparative Medicine
基金
山东省自然科学基金(ZR2021LZY041,ZR2022QH133)
国家自然科学基金项目(82174179,82204886)
中国博士后科学基金特别资助(2023T160398)
中国博士后科学基金(2021M702039)。
关键词
气虚湿瘀
肾纤维化
病证结合
动物模型
多组学
Qi deficiency and dampness stasis
renal fibrosis
combination of disease and syndrome
animal model
multiomics
作者简介
高冉冉(1998-),女,在读博士研究生,研究方向:中西医结合治疗肾系疾病的临床研究。E-mail:1377142543@qq.com;通信作者:李伟(1962-),女,博士,主任医师,研究方向:中西医结合治疗肾系疾病的临床研究。E-mail:lweidw@163.com。
