大黄附子汤改善小鼠重症急性胰腺炎肠动力障碍的机制研究

doi:10.3877/cma.j.issn.2095-9133.2022.04.005

中华卫生应急电子杂志 ›› 2022, Vol. 08 ›› Issue (04) : 216 -221. doi: 10.3877/cma.j.issn.2095-9133.2022.04.005

论著

大黄附子汤改善小鼠重症急性胰腺炎肠动力障碍的机制研究

宋轶¹, 仲益龙², 路晓光¹^,^†(

), 战丽彬³, 范治伟¹, 路童⁴, 陈海铭⁴, 芮庆林⁵

^1. 116001　辽宁大连，大连大学附属中山医院急诊医学科
^2. 116622　辽宁大连，大连大学研究生院
^3. 110847　辽宁沈阳，辽宁中医药大学中医药创新工程技术中心
^4. 110847　辽宁沈阳，辽宁中医药大学附属医院急诊医学科
^5. 210004　江苏南京，江苏省中医院急诊医学科

收稿日期:2022-04-18 出版日期:2022-08-18
通信作者: 路晓光
基金资助:
国家自然科学基金(81673801，81473512)

Mechanism of Dahuang Fuzi decoction in alleviating intestinal motility disorder in mice with severe acute pancreatitis

Yi Song¹, Yilong Zhong², Xiaoguang Lu¹^,^†(), Libin Zhan³, Zhiwei Fan¹, Tong Lu⁴, Haiming Chen⁴, Qinglin Rui⁵

^1. Department of Emergency Medicine, the Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China
^2. Graduate School of Dalian University, Dalian 116622, China
^3. Innovation Engineering and Technology Center of Traditional Chinese Medicine, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China
^4. Department of Emergency Medicine, the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China
^5. Department of Emergency Medicine, Jiangsu Hospital of Traditional Chinese Medicine, Nanjing 210004, China

Received:2022-04-18 Published:2022-08-18
Corresponding author: Xiaoguang Lu

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宋轶, 仲益龙, 路晓光, 战丽彬, 范治伟, 路童, 陈海铭, 芮庆林. 大黄附子汤改善小鼠重症急性胰腺炎肠动力障碍的机制研究[J]. 中华卫生应急电子杂志, 2022, 08(04): 216-221.

Yi Song, Yilong Zhong, Xiaoguang Lu, Libin Zhan, Zhiwei Fan, Tong Lu, Haiming Chen, Qinglin Rui. Mechanism of Dahuang Fuzi decoction in alleviating intestinal motility disorder in mice with severe acute pancreatitis[J]. Chinese Journal of Hygiene Rescue(Electronic Edition), 2022, 08(04): 216-221.

目的

揭示大黄附子汤改善重症急性胰腺炎小鼠早期肠动力障碍的机制。

方法

将27只SPF级雄性C57BL/6小鼠随机分为对照组、重症急性胰腺炎组(SAP)和大黄附子汤治疗组(DHFZT)，每组9只。SAP组和DHZFT组采用腹腔注射20% L-精氨酸溶液复制SAP小鼠模型，对照组腹腔注射等渗盐水。DHFZT组分别于造模后12、24、36 h以0.2 mL大黄附子汤灌肠，对照组和SAP组分别以0.2 mL等渗盐水灌肠。造模后48 h，每组取3只小鼠测定小肠推进率，其余小鼠麻醉后取血、取材。ELISA法检测小鼠血清淀粉酶、血钙、内毒素、TNF-α和IL-6含量。HE染色观察胰腺和回肠组织病理学变化。Western blot定量检测回肠nNOS、ChAT、VIP、SP蛋白表达。

结果

与对照组相比，SAP组的抑制性神经元nNOS和VIP数量显著增加，兴奋性神经元ChAT和SP数量显著下降，小肠推进率显著降低，对应血清淀粉酶、内毒素、TNF-α和IL-6水平显著升高，差异有统计学意义(P均<0.05)；与SAP组相比，DHFZT组的抑制性神经元nNOS和VIP数量显著下降，而兴奋性神经元ChAT和SP数量显著增加，小肠推进率显著增加，对应血清淀粉酶、内毒素、TNF-α和IL-6水平显著降低，血钙含量显著上升，差异有统计学意义(P均<0.05)。

结论

SAP小鼠早期发生肠动力障碍的机制可能存在肠神经系统的重塑，表现在抑制性神经元数量增加、兴奋性神经元数量减少，肠动力受抑制；大黄附子汤能够逆转肠神经系统的重塑，从而促进SAP小鼠肠动力的恢复。

关键词: 重症急性胰腺炎, 肠动力障碍, 肠神经系统, 大黄附子汤

Objective

To reveal the possible mechanism of intestinal motility disorder in mice with severe acute pancreatitis (SAP) and the effect of Dahuangfuzi decoction on it.

Methods

Twenty seven SPF male C57BL/6 mice were randomly divided into control group, severe acute pancreatitis group (SAP) and rhubarb and aconite decoction treatment group (DHFZT), with 9 mice in each group. SAP group and DHZFT group were intraperitoneally injected with 20% L-arginine solution to replicate SAP mouse model, and the control group was intraperitoneally injected with isotonic saline. In DHFZT group, 0.2 mL rhubarb and aconite decoction was used for enema 12, 24 and 36 hours after modeling, while 0.2 mL isotonic saline was used for enema in control group and SAP group. At 48h after modeling, 3 mice in each group were taken to measure the small intestine propulsion rate, and the rest of the mice were taken blood and materials after anesthesia. Detection of serum amylase, blood calcium, endotoxin and TNF in mice by ELISA- α And IL-6 content. Histopathological changes of pancreas and ileum were observed with HE staining. Western blot was used to detect the expression of nNOS, ChAT, VIP and SP proteins in ileum.

Results

Compared with the control group, the expression of nNOS and VIP in the SAP group were increased significantly, and the ChAT and SP were decreased significantly; the propulsion rate of small intestine was decreased significantly, with the lower levels of serum amylase, endotoxin, TNF-α and IL-6, and the blood calcium content also significantly decreased. Compared with the SAP group, the expression of nNOS and VIP in the DHFZT group were decreased significantly, and the ChAT and SP were increased significantly; the propulsion rate of small intestine was increased significantly, with the lower levels of serum amylase, endotoxin, TNF-α and IL-6, and the blood calcium content also significantly increased.

Conclusion

The mechanism of intestinal motility disorder in the early course of SAP mice may be related to the ENS remodeling, the increased number of inhibitory neurons, the decreased number of excitatory neurons, and therefore the proportion was imbalanced, leading to intestinal motility inhibition. Dahuangfuzi decoction may reverse the remodeling of ENS neurons and promote the recovery of intestinal motility in SAP mice.

Key words: Severe acute pancreatitis, Intestinal motility disorder, Enteric nervous system, Dahuangfuzi decoction

表1 各组小鼠血清淀粉酶、血钙含量(±s)

组别	鼠数	淀粉酶(U/L)	血钙(mmol/L)
对照组	6	215.7±6.8	2.36±0.35
SAP组	6	628.5±48.7^a	1.30±0.42^a
DHFZT组	6	453.5±28.8^b	1.91±0.14^b
F值		92.53	16.32
P值		<0.05	<0.05

表1 各组小鼠血清淀粉酶、血钙含量(±s)

组别	鼠数	淀粉酶(U/L)	血钙(mmol/L)
对照组	6	215.7±6.8	2.36±0.35
SAP组	6	628.5±48.7^a	1.30±0.42^a
DHFZT组	6	453.5±28.8^b	1.91±0.14^b
F值		92.53	16.32
P值		<0.05	<0.05

表2 血清内毒素、TNF-α和IL-6水平含量(±s)

组别	鼠数	内毒素(EU/L)	TNF-α(pg/mL)	IL-6(pg/mL)
对照组	6	592.78±40.89	236.9±7.82	54.9±4.01
SAP组	6	1144.68±63.84^a	705.6±35.24^a	216.9±8.21^a
DHFZT组	6	754.03±42.21^b	478.5±29.08^b	125.7±3.73^b
F值		151.56	16.73	26.36
P值		<0.05	<0.05	<0.05

表2 血清内毒素、TNF-α和IL-6水平含量(±s)

组别	鼠数	内毒素(EU/L)	TNF-α(pg/mL)	IL-6(pg/mL)
对照组	6	592.78±40.89	236.9±7.82	54.9±4.01
SAP组	6	1144.68±63.84^a	705.6±35.24^a	216.9±8.21^a
DHFZT组	6	754.03±42.21^b	478.5±29.08^b	125.7±3.73^b
F值		151.56	16.73	26.36
P值		<0.05	<0.05	<0.05

图1 小鼠胰腺、回肠HE染色(200×)注：SAP为重症胰腺炎组，DHFZT为大黄附子汤治疗组

图2 Western blot检测各组小鼠回肠组织nNOS、ChAT、VIP、SP蛋白表达注：nNOS为一氧化氮合酶，ChAT为胆碱乙酰转移酶，VIP为血管活性肠肽，SP为P物质；SAP为重症胰腺炎组，DHFZT为大黄附子汤治疗组；SAP组与对照组相比：^aP<0.05，治疗组与SAP组相比：^bP<0.05

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