巯基丙酮酸硫基转移酶调控核因子κB信号介导自噬对重症急性胰腺炎大鼠的影响及机制

doi:10.3877/cma.j.issn.2095-2015.2023.06.012

中华消化病与影像杂志(电子版) ›› 2023, Vol. 13 ›› Issue (06) : 422 -426. doi: 10.3877/cma.j.issn.2095-2015.2023.06.012

论著

巯基丙酮酸硫基转移酶调控核因子κB信号介导自噬对重症急性胰腺炎大鼠的影响及机制

王小红, 钱晶, 翁文俊, 周国雄, 朱顺星, 祁小鸣, 刘春, 王萍, 沈伟, 程睿智, 秦璟灏^†(

)

211900　江苏仪征，南京鼓楼医院集团仪征医院消化内科
211900　江苏仪征，南京鼓楼医院集团仪征医院普通外科
211900　江苏仪征，南京鼓楼医院集团仪征医院心胸外科
226001　江苏省，南通大学附属医院消化内科
226001　江苏省，南通大学实验动物中心
213000　江苏常州，苏州大学附属第三医院中医科

收稿日期:2023-03-06 出版日期:2023-12-01
通信作者: 秦璟灏
基金资助:
扬州市重点研发项目（社会发展）(YZ2021091)

Effects of regulation of nuclear factor-κB signal by mercaptopyruvate sulfurtransferase mediated autophagy on rats with severe acute pancreatitis and its mechanism

Xiaohong Wang, Jing Qian, Wenjun Weng, Guoxiong Zhou, Shunxing Zhu, Xiaoming Qi, Chun Liu, Ping Wang, Wei Shen, Ruizhi Cheng, Jinghao Qin^†()

Department of Gastroenterology, Yizheng Hospital of Nanjing Drum Tower Hospital Group, Yizheng 211900, China
Department of General Surgery, Yizheng Hospital of Nanjing Drum Tower Hospital Group, Yizheng 211900, China
Department of Cardiothoracic Surgery, Yizheng Hospital of Nanjing Drum Tower Hospital Group, Yizheng 211900, China
Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, China
Laboratory Animal Center, Nantong University, Nantong 226001, China
Department of Traditional Chinese Medicine, Third Affiliated Hospital of Soochow University, Changzhou 213000, China

Received:2023-03-06 Published:2023-12-01
Corresponding author: Jinghao Qin

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引用本文：

王小红, 钱晶, 翁文俊, 周国雄, 朱顺星, 祁小鸣, 刘春, 王萍, 沈伟, 程睿智, 秦璟灏. 巯基丙酮酸硫基转移酶调控核因子κB信号介导自噬对重症急性胰腺炎大鼠的影响及机制[J/OL]. 中华消化病与影像杂志(电子版), 2023, 13(06): 422-426.

Xiaohong Wang, Jing Qian, Wenjun Weng, Guoxiong Zhou, Shunxing Zhu, Xiaoming Qi, Chun Liu, Ping Wang, Wei Shen, Ruizhi Cheng, Jinghao Qin. Effects of regulation of nuclear factor-κB signal by mercaptopyruvate sulfurtransferase mediated autophagy on rats with severe acute pancreatitis and its mechanism[J/OL]. Chinese Journal of Digestion and Medical Imageology(Electronic Edition), 2023, 13(06): 422-426.

目的

研究巯基丙酮酸硫基转移酶(MPST)调控核因子κB(NF-κB)信号介导自噬对重症急性胰腺炎(SAP)大鼠的影响。

方法

将50只SD大鼠随机分为Sham组、SAP组(重症急性胰腺炎造模)、SAP＋AAV-NC组(AAV对照病毒注射＋ SAP造模)、SAP＋AAV-MPST OE组(AAV MPST过表达病毒注射＋ SAP造模)和SAP＋AAV-MPST shRNA组(AAV MPST敲除病毒注射＋ SAP造模)。分别采用HE染色和胰腺组织损伤评分评价胰腺炎病情严重程度；电镜观察胰腺组织自噬泡；比色法检测血清淀粉酶和脂肪酶的活性；ELISA法检测胰腺组织IL-6表达水平；Western blot检测胰腺组织MPST、LC3 Ⅱ/Ⅰ、beclin1、ATG5、p-NF-κB、NF-κB、β-actin蛋白表达水平。

结果

与SAP组相比，SAP＋AAV-MPST OE组大鼠胰腺损伤评分、自噬泡数量、血清淀粉酶和脂肪酶活性水平、IL-6水平、MPST、LC3 Ⅱ/Ⅰ、beclin1、ATG5和p-NF-κB/NF-κB水平显著升高，而SAP＋ AAV-MPST shRNA组大鼠这些指标均显著降低(P<0.01)。

结论

MPST可通过NF-κB信号诱导胰腺组织自噬和炎症反应，进而促进SAP病情；抑制MPST对SAP的治疗具有重要意义。

关键词: 重症急性胰腺炎, 巯基丙酮酸硫基转移酶, 自噬, 核因子κB, 大鼠，SD

Objective

To study the effects of regulation of nuclear factor-κB(NF-κB)signal by mercaptopyruvate sulfurtransferase(MPST)mediated autophagy on rats with severe acute pancreatitis(SAP).

Methods

Fifty SD rats were randomly divided into Sham group, SAP group(severe acute pancreatitis model), SAP+ AAV-NC group(AAV control virus injection+ SAP model), SAP+ AAV-MPST OE group(AAV MPST over-expression virus injection+ SAP model), and SAP+ AAV-MPST shRNA group(AAV MPST knockout virus injection+ SAP model). The severity of pancreatitis using HE staining and pancreatic tissue injury score were evaluated; Autophagic vesicles in pancreatic tissue were observed using electron microscopy; The activity of serum amylase and lipase was detected using colorimetric method; IL-6 levels in pancreatic tissue was detected using ELISA; The expression levels of MPST, LC3 Ⅱ/Ⅰ, beclin1, ATG5, p-NF-κB, NF-κB and β-actin were evaluated by Western blot.

Results

Compared with the SAP group, the pancreatic injury score, number of autophagic vesicles, serum amylase and lipase activity levels, IL-6 level, MPST, LC3 Ⅱ/Ⅰ, beclin1, ATG5, and p-NF-κB/NF-κB levels were significantly increased in SAP+ AAV-MPST OE group, while the SAP+ AAV-MPST shRNA group showed a significant decrease in these indicators(P<0.01).

Conclusion

MPST can induce autophagy and inflammatory response in pancreatic tissue through NF-κB signal, thereby promoting the condition of SAP.Inhibiting MPST is of great significance for the treatment of SAP.

Key words: Severe acute pancreatitis, Mercaptopyruvate sulfurtransferase, Autophagy, Nuclear factor-κB, Rat, SD

图1 各组大鼠胰腺组织病理学形态(HE染色，×400)注：1A为Sham组；1B为SAP组；1C为SAP＋AAV－NC组；1D为SAP＋AAV－MPST OE组；1E为SAP＋AAV－MPST shRNA组。

图2 电镜观测各组大鼠胰腺组织的自噬泡数量注：2A为Sham组；2B为SAP组；2C为SAP＋AAV－NC组；2D为SAP＋AAV－MPST OE组；2E为SAP＋AAV－MPST shRNA组。箭头所指为自噬泡。

表1 各组大鼠自噬泡数量和胰腺损伤评分的比较(±s)

组别	鼠数	自噬泡数量(个)	胰腺损伤评分(分)
Sham组	10	0.60±0.52^b	0.70±0.67^b
SAP组	8	5.13±1.13^a	10.13±1.13^a
SAP＋AAV-NC组	8	4.88±0.83^a	9.88±0.64^a
SAP＋AAV-MPST OE组	7	8.86±1.35^ab	14.57±1.51^ab
SAP＋AAV-MPST shRNA组	9	2.22±0.83^ab	6.11±1.27^ab
F		92.45	202.23
P		<0.0001	<0.0001

表1 各组大鼠自噬泡数量和胰腺损伤评分的比较(±s)

组别	鼠数	自噬泡数量(个)	胰腺损伤评分(分)
Sham组	10	0.60±0.52^b	0.70±0.67^b
SAP组	8	5.13±1.13^a	10.13±1.13^a
SAP＋AAV-NC组	8	4.88±0.83^a	9.88±0.64^a
SAP＋AAV-MPST OE组	7	8.86±1.35^ab	14.57±1.51^ab
SAP＋AAV-MPST shRNA组	9	2.22±0.83^ab	6.11±1.27^ab
F		92.45	202.23
P		<0.0001	<0.0001

表2 各组大鼠血清淀粉酶和脂肪酶活性及胰腺组织炎症因子IL-6表达水平的比较(±s)

组别	鼠数	淀粉酶(U/L)	脂肪酶(U/L)	IL-6(pg/mL)
Sham组	10	3061.30±382.85^b	28.10±3.41^b	67.32±9.94^b
SAP组	8	10 066.75±1666.02^a	246.50±49.52^a	174.70±15.09^a
SAP＋AAV-NC组	8	9901.38±1203.67^a	239.63±39.98^a	167.49±11.22^ab
SAP＋AAV-MPST OE组	7	14 509.86±1141.70^ab	338.43±16.82^ab	287.03±20.01^ab
SAP＋AAV-MPST shRNA组	9	6515.56±601.04^ab	115.56±26.55^ab	97.56±13.81^ab
F		137.51	131.18	294.07
P		<0.0001	<0.0001	<0.0001

表2 各组大鼠血清淀粉酶和脂肪酶活性及胰腺组织炎症因子IL-6表达水平的比较(±s)

组别	鼠数	淀粉酶(U/L)	脂肪酶(U/L)	IL-6(pg/mL)
Sham组	10	3061.30±382.85^b	28.10±3.41^b	67.32±9.94^b
SAP组	8	10 066.75±1666.02^a	246.50±49.52^a	174.70±15.09^a
SAP＋AAV-NC组	8	9901.38±1203.67^a	239.63±39.98^a	167.49±11.22^ab
SAP＋AAV-MPST OE组	7	14 509.86±1141.70^ab	338.43±16.82^ab	287.03±20.01^ab
SAP＋AAV-MPST shRNA组	9	6515.56±601.04^ab	115.56±26.55^ab	97.56±13.81^ab
F		137.51	131.18	294.07
P		<0.0001	<0.0001	<0.0001

图3 各组大鼠胰腺组织MPST、自噬相关分子及NF-κB蛋白表达典型Western blot条图注：A为Sham组；B为SAP组；C为SAP＋AAV－NC组；D为SAP＋AAV－MPST shRNA组；E为SAP＋AAV－MPST OE组。

表3 各组大鼠胰腺组织MPST、LC3 Ⅱ/Ⅰ、beclin1、ATG5和p-NF-κB/NF-κB蛋白表达水平的比较(±s)

组别	鼠数	MPST/β-actin	LC3 Ⅱ/Ⅰ /β-actin	beclin1/β-actin	ATG5/β-actin	p-NF-κB/NF-κB
Sham组	10	1.19±0.21^b	1.03±0.08^b	0.98±0.13^b	1.15±0.12^b	1.06±0.11^b
SAP组	8	2.55±0.31^a	3.02±0.21^a	1.85±0.17^a	2.09±0.17^a	2.61±0.15^a
SAP＋AAV-NC组	8	2.45±0.29^a	2.90±0.27^a	1.80±0.13^a	1.99±0.15^a	2.52±0.14^a
SAP＋AAV-MPST shRNA组	9	1.78±0.21^ab	1.58±0.15^ab	1.43±0.21^ab	1.53±0.18^ab	1.88±0.16^ab
SAP＋AAV-MPST OE组	7	3.39±0.30^ab	3.65±0.22^ab	3.15±0.27^ab	3.06±0.21^ab	3.26±0.22^ab
F		83.70	276.50	149.90	150.11	253.39
P		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

表3 各组大鼠胰腺组织MPST、LC3 Ⅱ/Ⅰ、beclin1、ATG5和p-NF-κB/NF-κB蛋白表达水平的比较(±s)

组别	鼠数	MPST/β-actin	LC3 Ⅱ/Ⅰ /β-actin	beclin1/β-actin	ATG5/β-actin	p-NF-κB/NF-κB
Sham组	10	1.19±0.21^b	1.03±0.08^b	0.98±0.13^b	1.15±0.12^b	1.06±0.11^b
SAP组	8	2.55±0.31^a	3.02±0.21^a	1.85±0.17^a	2.09±0.17^a	2.61±0.15^a
SAP＋AAV-NC组	8	2.45±0.29^a	2.90±0.27^a	1.80±0.13^a	1.99±0.15^a	2.52±0.14^a
SAP＋AAV-MPST shRNA组	9	1.78±0.21^ab	1.58±0.15^ab	1.43±0.21^ab	1.53±0.18^ab	1.88±0.16^ab
SAP＋AAV-MPST OE组	7	3.39±0.30^ab	3.65±0.22^ab	3.15±0.27^ab	3.06±0.21^ab	3.26±0.22^ab
F		83.70	276.50	149.90	150.11	253.39
P		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

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Effects of regulation of nuclear factor-κB signal by mercaptopyruvate sulfurtransferase mediated autophagy on rats with severe acute pancreatitis and its mechanism