金丝桃素通过调控NLRP3炎症小体改善小鼠急性胰腺炎的机制探讨

doi:10.11958/20251147

摘要/Abstract

摘要：

目的探究金丝桃素（HY）对小鼠急性胰腺炎（AP）的治疗作用，并揭示其对NOD样受体家族蛋白3（NLRP3）炎症小体信号通路的影响。方法以雨蛙素（CER）建立小鼠AP模型，通过随机抽样的方式分为正常对照组、模型组、HY低剂量（CER+HY 5 mg/kg）组、HY中剂量（CER+HY 10 mg/kg）组、HY高剂量（CER+HY 20 mg/kg）组，每组10只。小鼠266-6胰腺腺泡癌细胞利用胆囊收缩素（CCK）处理并分为Control组、AP组、CCK+HY 1 μmol/L组、CCK+HY 2 μmol/L组、CCK+HY 4 μmol/L组。采用酶联免疫吸附试验（ELISA）检测各组小鼠血清淀粉酶（AMS）、脂肪酶、胰蛋白酶和髓过氧化物酶（MPO）的活性，检测炎性因子白细胞介素（IL）-1β和肿瘤坏死因子（TNF）-α水平；Western blot检测NLRP3表达；实时定量PCR（qPCR）检测各组小鼠胰腺组织中NLRP3、胱天蛋白酶（Caspase）-1、IL-1β、TNF-α及IL-18 mRNA水平；CCK8法检测各组细胞存活率；qPCR检测各组细胞中NLRP3、Caspase-1和IL-18 mRNA表达水平。结果与正常对照组相比，模型组小鼠血清中AMS、脂肪酶、胰蛋白酶、MPO、IL-1β和TNF-α水平升高，胰腺组织中NLRP3 mRNA及蛋白表达水平，IL-1β和TNF-α、IL-18和Caspase-1 mRNA水平升高（P<0.01）；与模型组相比，HY低、中、高剂量组血清AMS、IL-1β和TNF-α的水平，胰蛋白酶活性，胰腺组织中IL-1β、TNF-α、IL-18、Caspase-1 mRNA水平均降低，HY中剂量组和HY高剂量组血清脂肪酶、MPO水平，胰腺组织NLRP3 mRNA及蛋白表达降低（P<0.05）。与AP组比较，CCK+HY 1 μmol/L组、CCK+HY 2 μmol/L组、CCK+HY 4 μmol/L组细胞存活率均升高，NLRP3、IL-18和Caspase-1 mRNA水平降低，并呈剂量依赖性（P<0.05）。结论金丝桃素在体内外均能有效治疗急性胰腺炎，其发挥治疗作用可能与调控NLRP3炎症小体信号通路有关。

关键词: 胰腺炎, 金丝桃素, NLR家族, 热蛋白结构域包含蛋白3, 炎症

Abstract:

Objective To investigate the therapeutic effect of hypericin on acute pancreatitis (AP) in mice and its effect on NLRP3 inflammasome signaling pathway. Methods The AP model in mice was established with caerulein (CER). The mice were divided into the normal control group, the model group (AP group), the low-dose HY group (CER + HY 5 mg/kg group), the medium-dose HY group (CER + HY 10 mg/kg group) and the high-dose HY group (CER + HY 20 mg/kg group), with 10 mice in each group. The 266-6 mouse pancreatic acinar cancer cells were treated with cholecystokinin (CCK) and divided into the control group, the AP group, the CCK + HY 1 μmol/L group, the CCK + HY 2 μmol/L group and the CCK + HY 4 μmol/L group. The activities of amylase (AMS), lipase, trypsin and myeloperoxidase (MPO) in the serum of each group of mice, and levels of inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA). The expression of NOD-like receptor family protein 3 (NLRP3) was detected by Western blot assay. The mRNA levels of NLRP3, caspase (Caspase)-1, IL-1β, TNF-α and IL-18 in pancreatic tissue of mice were detected by real-time quantitative PCR (q-PCR). The cell survival rate of cells in each group was detected by CCK8 method. The mRNA expression levels of NLRP3, Caspase-1 and IL-18 in each group of cells were detected by q-PCR. Results Compared with the normal control group, the levels of AMS, lipase, MPO, trypsin, IL-1β and TNF-α in serum of the model group, and the mRNA and protein expression levels of NLRP3, IL-1β, TNF-α, IL-18 and Caspase-1 in pancreatic tissue were increased (P<0.01). Compared with the model group, the levels of AMS, IL-1β and TNF-α, the enzymatic activity of trypsin in serum, and the mRNA levels of IL-1β, TNF-α, IL-18 and Caspase-1 in pancreatic tissue were decreased in the low-, medium- and high-dose HY groups. The serum levels of lipase and MPO and the mRNA expression levels of NLRP3 in pancreatic tissue were decreased in the medium- and high-dose HY groups (P<0.05). Compared with the AP group, the cell survival rates were increased in the CCK + HY 1 μmol/L group, the CCK + HY 2 μmol/L group and the CCK + HY 4 μmol/L group, and the mRNA levels of NLRP3, IL-18 and Caspase-1 were decreased in a dose-dependent manner (P<0.05). Conclusion Hypericin can effectively treat AP in vivo and in vitro, and its therapeutic effect may be related to the regulation of NLRP3 inflammasome signaling pathway.

Key words: pancreatitis, Hypericin, NLR family, pyrin domain-containing 3 protein, inflammation

中图分类号:

R576.1

图/表 7

参考文献 27

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基因名称	引物序列（5'→3'）	产物大小/bp
β-actin	上游：AGAGGGAAATCGTGCGTGAC	236
β-actin	下游：CAATAGTGATGACCTGGCCGT	236
NLRP3	上游：TCCACAATTCTGACCCACAA	228
NLRP3	下游：ACCTCACAGAGGGTCACCAC	228
Caspase-1	上游：GGGCCCCAGGCAAGCCAAATC	225
Caspase-1	下游：AGGGCAAGACGTGTACGAGTGGT	225
IL-1β	上游：TCTTTGAAGTTGACGGACCC	235
IL-1β	下游：TGAGTGATACTG CCTGCCTG	235
TNF-α	上游：GTCGCTACCGTCGTGACTTC	216
TNF-α	下游：CAGACATGCACCTACCCAGC	216
IL-18	上游：ACACCAGCCTGGCTTCCATC	208
IL-18	下游：TTGGAGCTGGAGCTGCTTATAGTTG	208

基因名称	引物序列（5'→3'）	产物大小/bp
β-actin	上游：AGAGGGAAATCGTGCGTGAC	236
β-actin	下游：CAATAGTGATGACCTGGCCGT	236
NLRP3	上游：TCCACAATTCTGACCCACAA	228
NLRP3	下游：ACCTCACAGAGGGTCACCAC	228
Caspase-1	上游：GGGCCCCAGGCAAGCCAAATC	225
Caspase-1	下游：AGGGCAAGACGTGTACGAGTGGT	225
IL-1β	上游：TCTTTGAAGTTGACGGACCC	235
IL-1β	下游：TGAGTGATACTG CCTGCCTG	235
TNF-α	上游：GTCGCTACCGTCGTGACTTC	216
TNF-α	下游：CAGACATGCACCTACCCAGC	216
IL-18	上游：ACACCAGCCTGGCTTCCATC	208
IL-18	下游：TTGGAGCTGGAGCTGCTTATAGTTG	208

组别	AMS/（U/L）	脂肪酶/（U/L）
正常对照组	1 957.14±23.80	189.92±13.03
模型组	6 512.55±100.10^a	383.03±29.85^a
HY低剂量组	5 031.68±43.70^b	348.75±28.77
HY中剂量组	4 137.10±40.90^bc	310.43±22.43^bc
HY高剂量组	2 129.28±31.20^bcd	231.32±17.65^bcd
F	399.100^＊＊	82.500^＊＊

组别	AMS/（U/L）	脂肪酶/（U/L）
正常对照组	1 957.14±23.80	189.92±13.03
模型组	6 512.55±100.10^a	383.03±29.85^a
HY低剂量组	5 031.68±43.70^b	348.75±28.77
HY中剂量组	4 137.10±40.90^bc	310.43±22.43^bc
HY高剂量组	2 129.28±31.20^bcd	231.32±17.65^bcd
F	399.100^＊＊	82.500^＊＊

组别	胰蛋白酶/（U/mg）	MPO/（U/g）
正常对照组	4.59±0.33	1.25±0.08
模型组	15.22±1.05^a	2.77±0.36^a
HY低剂量组	11.28±0.75^b	2.61±0.39
HY中剂量组	8.38±0.66^b	2.33±0.26^bc
HY高剂量组	5.23±0.34^bcd	1.41±0.11^bcd
F	209.900^＊＊	45.550^＊＊