洋甘菊总黄酮通过调控NLRP3/ NF-κ B炎症通路改善db/db 2型糖尿病小鼠肝损伤

Total chamomile flavonoids ameliorate liver injury in db/db mice by modulating the NLRP3/ NF-κ B inflammatory pathway

关键词:  洋甘菊总黄酮  db/db小鼠  肝损伤  NLRP3/ NF-κB炎症通路。

Key words:  [Keywords] Chamomile total flavonoids  db/db mice  liver injury  NLRP3/ NF-κ B inflammatory pathway.

摘要:

[摘要]目的：探究洋甘菊总黄酮是否通过NOD 样受体蛋白 3（NOD-like receptor family pyrin domain containing 3，NLRP3）/核转录因子-κ B（nuclear factor kappa-B， NF-κ B）通路减轻2型糖尿病 （type 2 diabetes mellitus，T2DM）db/db 小鼠肝损伤。方法：HPLC法同时测定洋甘菊总黄酮中槲皮素、木犀草素、芹菜素含量；选取 SPF 级db/db 小鼠 40只及对照db/m 小鼠 8只，适应性喂养 7d后检测血糖，随机分为 6 组，每组 8只，正常组、模型组、二甲双胍组、洋甘菊总黄酮低剂量组、中剂量、高剂量组，连续灌胃给药7周，每日观察小鼠一般情况。每周记录小鼠体重，每周使用血糖仪检测每组小鼠FBG。检测每组小鼠血清中高密度脂蛋白胆固醇（High density lipoprotein cholesterol，HDL-C）、低密度脂蛋白胆固醇（Low-Density Lipoprotein Cholesterol，LDL-C）、甘油三酯（Triglyceride，TG、总胆固醇（Total Cholesterol，TC）、谷丙转氨酶（glutamic pyruvic transaminase， ALT），谷草转氨酶（glutamic oxaloacetic transaminase， AST）、白细胞介素-6(interleukin-6 ,IL-6)、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的含量。测定每组小鼠肝组织中丙二醛 （Malondialdehyde，MDA）表达量、超氧化物歧化酶（Superoxide dismutase，SOD）及 谷胱甘肽过氧化物酶（Glutathione peroxidase，GSH-Px） 酶的活性。Western blot检测每组小鼠肝组织 NLRP3、NF-κB、C-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK）、白细胞介素-1β(interleukin-1β)的蛋白表达情况以及RT-qPCR检测mRNA表达情况。结果: HPLC结果表明，测得供试品中槲皮素含量平均值为3.8218 mg·g-1，木犀草素含量平均值为5.119 mg·g-1，芹菜素含量平均值为3.5353 mg·g-1；与正常组比较，模型组小鼠体重、FBG、TG、TC、 LDL-C 表达水平明显升高（P<0.01）；HDL-C含量有明显的降低（P＜0.01）；肝组织中 MDA 、SOD表达显著升高（P<0.01），SOD 和 GSH-PX 酶活性显著降低（P<0.01）；肝脏组织固缩，肝脏内细胞空泡样变性，伴有炎性细胞浸润肝损伤严重。IL-6、TNF-α水平增加（P<0.01），肝脏组织 NLRP3、NF-κB、JNK、IL-1β 蛋白与mRNA基因表达水平均显著升高（P<0.01）。药物干预 7周后，与模型组比较，药物组小鼠体重、FBG、TG、TC、 LDL-C 表达水平降低（P<0.01），HDL-C含量有明显的降低（P＜0.01），肝组织中 MDA 表达显著降低（P<0.01），SOD 和 GSH-PX 酶活性显著升高（P<0.01），IL-6、TNF-α水平降低（P<0.01）、肝组织病变程度得到明显改善，肝组织 NLRP3、NF-κB、JNK、IL-1β蛋白与mRNA基因表达水平均降低（P<0.01）结论：洋甘菊总黄酮可以改善db/db小鼠的糖脂代谢，改善肝组织损伤，其机制可能通过调控 NLRP3/NF-κB信号通路，抑制细胞凋亡，减轻炎症反应，进而延缓糖尿病肝脏损伤的疾病进程。

Abstract:

ABSTRACT: OBJECTIVE To explore whether chamomile flavonoids can alleviate liver injury in type 2 diabetes db/db mice through the NOD like receptor protein 3 (NLRP3)/nuclear factor kappa B (NF - κ B) pathway. METHODS: Simultaneous determination of quercetin, luteolin, and apigenin content in total flavonoids of chamomile by HPLC method;Forty SPF grade db/db mice and eight control db/m mice were selected, and their blood glucose levels were measured after adaptive feeding for 7 days. They were randomly divided into six groups, with 8 mice in each group: normal group, model group, metformin group, low-dose group, medium dose group, and high-dose group of chamomile total flavonoids. The mice were orally administered continuously for 7 weeks, and their general conditions were observed daily. Record the weight of mice every week and use a blood glucose meter to measure the FBG of each group of mice every week. Detect HDL-C、LDL-C、TG、TC、ALT、AST、The levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF - α). Measure the expression level of MDA and the activity of SOD and GSH-PX enzymes in the liver tissue of each group of mice. Western blot was used to detect the protein expression of NLRP3, NF - κ B, JNK, and IL-1 β in the liver tissue of each group of mice, and RT qPCR was used to detect mRNA expression. RESULTS: The HPLC results showed that the average content of quercetin in the test sample was 3.8218 mg·g-1, the average content of luteolin was 5.119 mg·g-1, and the average content of apigenin was 3.5353 mg·g-1, Compared with the normal group, the body weight, FBG, TG, TC, and LDL-C expression levels of the model group mice were significantly increased (P<0.01); The HDL-C content showed a significant decrease (P<0.01); The expression of MDA in liver tissue significantly increased (P<0.01), while the activities of SOD and GSH-PX enzymes significantly decreased (P<0.01); Liver tissue consolidation, vacuolar degeneration of liver cells, accompanied by inflammatory cell infiltration, severe liver injury. The levels of IL-6 and TNF - α increased, and the protein and mRNA gene expression levels of NLRP3, NF - κ B, JNK, IL-1 β in liver tissue were significantly increased (P<0.01). After 7 weeks of drug intervention, compared with the model group, the weight, FBG, TG, TC, LDL-C expression levels of mice in the drug group decreased (P<0.01), HDL-C content significantly decreased (P<0.01), MDA expression in liver tissue significantly decreased (P<0.01), SOD and GSH-PX enzyme activity significantly increased (P<0.01), IL-6, TNF - α levels decreased (P<0.01),, the degree of liver lesions significantly improved, and the expression levels of NLRP3, NF - κB, JNK, IL-1 β protein and mRNA gene in liver tissue decreased (P<0.01) . CONCLUSION Chamomile flavonoids can improve the metabolism of glucose and lipid in db/db mice and improve the liver The mechanism of tissue damage may be through regulating NLRP3/NF - κ B signaling pathway, inhibiting cell apoptosis, reducing inflammatory reaction, and then delaying the disease process of diabetes liver injury.