小陷胸汤加味方对2型糖尿病大鼠肝损伤的保护作用及其机制

doi:10.13481/j.1671-587X.20230308

摘要/Abstract

摘要：

目的探讨小陷胸汤加味方（MXD）对2型糖尿病（T2DM）大鼠肝损伤的保护作用，阐明其可能的作用机制。方法 50只大鼠采用高糖高脂饲料喂养并联合链脲佐菌素（STZ）腹腔注射构建T2DM大鼠模型，40只造模成功大鼠随机分为模型组（不给予药物）、二甲双胍组（给予200 mg·kg^－1二甲双胍）、低剂量MXD组（给予630 mg·kg^－1 MXD）和高剂量MXD组（给予1 260 mg·kg^－1 MXD），每组10只；另取10只大鼠作为对照组（给予标准饲料）。称量各组大鼠体质量，检测各组大鼠空腹血糖（FBG）水平，全自动生化分析仪检测各组大鼠血清中丙氨酸氨基转氨酶（ALT）和天冬氨酸氨基转氨酶（AST）活性，HE染色观察各组大鼠肝组织病理形态表现，酶联免疫吸附测定（ELISA）法检测各组大鼠血清中白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）水平，分光光度计检测各组大鼠肝组织中超氧化物歧化酶（SOD）活性和还原型谷胱甘肽（GSH）及丙二醛（MDA）水平，Western blotting法检测各组大鼠肝组织中核因子E2相关因子2（Nrf2）和血红素加氧酶（HO-1）蛋白表达水平。结果对照组大鼠摄食和摄水量适中，均无骤增和骤减情况；模型组大鼠在注射STZ溶液后出现糖尿病的典型症状；与模型组比较，二甲双胍组、低剂量MXD组和高剂量MXD组大鼠糖尿病症状均有不同程度改善。与对照组比较，各时间点模型组大鼠体质量明显减小（P<0.01），FBG水平均明显升高（P<0.01）；与模型组比较，给药第2、3和4周，二甲双胍组和高剂量MXD组大鼠FBG水平明显降低（P<0.01）；给药第3和4周，低剂量MXD组大鼠FBG水平明显降低（P<0.05或P<0.01）。与对照组比较，模型组大鼠血清中AST和ALT活性均明显升高（P<0.01）；与模型组比较，二甲双胍组、低剂量MXD组和高剂量MXD组大鼠血清中ALT和AST活性明显降低（P<0.01）。HE染色，对照组大鼠肝细胞形态正常，未见炎症细胞浸润；模型组可见严重的炎症细胞浸润状态；二甲双胍组可见少量炎症细胞浸润；低和高剂量MXD组炎症细胞浸润现象基本消失。ELISA法检测，与对照组比较，模型组大鼠血清中IL-1β、IL-6和TNF-α水平均明显升高（P<0.01）；与模型组比较，二甲双胍组、低剂量MXD组和高剂量MXD组大鼠血清中IL-1β、IL-6和TNF-α水平均明显降低（P<0.01）。分光光度计检测，与对照组比较，模型组大鼠肝组织中SOD活性和GSH水平明显降低（P<0.01），MDA水平明显升高（P<0.01）；与模型组比较，二甲双胍组、低剂量MXD组和高剂量MXD组大鼠肝组织中SOD活性和GSH水平均明显升高（P<0.01），低和高剂量MXD组大鼠肝组织中MDA水平明显降低（P<0.05或P<0.01）。Western blotting法检测，与对照组比较，模型组大鼠肝组织中Nrf2和 HO-1蛋白表达水平明显降低（P<0.01）；与模型组比较，二甲双胍组、低剂量MXD组和高剂量MXD组大鼠肝组织中Nrf2和HO-1蛋白表达水平均明显升高（P<0.05或P<0.01）。结论 MXD对T2DM大鼠肝损伤有保护作用，其机制可能与抗炎及活化Nrf2/HO-1信号通路进而抑制氧化应激有关。

关键词: 小陷胸汤加味方, 2型糖尿病, 肝损伤, 炎症反应, 氧化应激

Abstract:

Objective To discuss the protective effect of Modified Xiao-Xian-Xiong Decoction （MXD） on liver injury in the rats with type 2 diabetes mellitus（T2DM）， and to clarify its possible mechanism. Methods Fifty rats were fed with high sugar and high fat combined with intraperitoneal injection of streptozotocin （STZ） to establish the T2DM rat models.A total of 40 rats successful modeling were randomly divided into model group（given no drugs），metformin group（given 200 mg·kg^－1 metformin）， low dose of MXD group （given 630 mg·kg^－1 MXD），and high dose of MXD group（given 1 260 mg·kg^－1 MXD）， and there were 10 rats in each group； another 10 rats were selected as control group（given standard feed）. The body masses of the rats in various groups were detected；the fasting blood glucose （FBG） levels of the rats in various groups were detected； the activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum of the rats in various groups were detected by fully automated biochemical analyzer；the pathomorphology of liver tissue of the rats in various groups were detected by HE staining； enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of interleukin 1β （IL-1β）， interleukin 6 （IL-6）， tumor necrosis factor α （TNF-α） in serum； the levels of superoxide dismutase （SOD）， reductive glutathione （GSH）， and malondialdehyde （MDA） in liver tissue of the rats in various groups were measured by spectrophotometer；the expression levels of nuclear factor E2 related factor 2 （Nrf2） and heme oxygenase （HO-1） proteins in liver tissue of the rats in various groups were detected by Western blotting method. Results The rats in control group had moderate feeding and water intake，and the above indexes of the rats had no sudden increasing and decreasing；the rats in model group had typical symptoms of diabetes；compared with model group，the diabetes symptoms of the rats in meftormin group，low dose of MXD group，and high dose of MXD group had been improved to various degrees.Compared with control group， the body masses of the rats in model group were significantly decreased at different time points（P<0.01）， while the FBG levels were increased （P<0.01）； compared wtih model group，the levels of FBG of the rats in meftormin group，low dose of MXD group，and high dose of MXD group were decreased in the 2nd，3rd and 4th weeks after administration，and the levels of FBG of the rats in low dose of MXD group were decreased in the 3rd and 4th weeks after administration（P<0.05 or P<0.01）.Compared with control group，the activities of AST and ALT in serum of the rats in model group were increased（P<0.01）；compared with model group，the activities of AST and ALT in serum of the rats in metformin group，low dose of MXD group，and high dose of MXD group were decreased（P<0.01）.The HE staining results showed that the morphology of liver cells of the rats in control group was normal，and there was no inflammatory cell infiltration；there was serious inflammatory cell infiltration in model group；there was a bit of inflammatory cell infiltration in meftormin group；there were almost no inflammatory cell infiltrations in low and high doses of MXD groups.The ELISA method results showed that the levels of serum IL-1β， IL-6， and TNF- α in serum of the rats in model group were significantly increased （P<0.01）.Compared with model group， the levels of serum IL-1β， IL-6， and TNF-α of the rats in metformin group， low dose of MXD and high dose of MXD groups were decreased（P<0.01）， while the activity of SOD， the level of GSH， and the expression levels of Nrf2 and HO-1 proteins in liver tissue of the rats in model group were significantly decreased （P<0.01）.The spectrophotometer results showed that compared with control group， the activity of SOD， the level of GSH in liver tissue of the rats in model group were decreased（P<0.01）， and the level of MDA was significantly increased （P<0.01）；compared with model group，the activities of SOD and the levels of GSH in liver tissue of the rats in metformin group，low dose of MXD group，and high dose of MXD group were decreased（P<0.01），and the levels of MDA in liver tissue of the rats in low and high doses of MXD groups were decreased（P<0.05 or P<0.01）.The Western blotting results showed that compared with control group，the expression levels of Nrf2 and HO-1 proteins in liver tissue of the rats in model group were decreased（P<0.01）；compared with model group， the expression levels of Nrf2 and HO-1 proteins in liver tissue of the rats in metformin group，low dose of MXD group，and high dose of MXD group were increased（P<0.05 or P<0.01）. Conclusion MXD has protective effect on liver injury of the rats with type 2 diabetes， and its mechanism may be related to anti-inflammation and activation of Nrf2/HO-1 signaling pathway，thereby inhibition of oxidative stress.

Key words: Modified Xiao-Xian-Xiong decoction, Type 2 diabetes mellitus, Liver injury, Inflammatory response, Oxidative stress

中图分类号:

R285.5

关敬,哈森,袁颢,陈颖,刘鹏举,刘智,姜爽. 小陷胸汤加味方对2型糖尿病大鼠肝损伤的保护作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(3): 608-616.

Jing GUAN,Shen HA,Hao YUAN,Ying CHEN,Pengju LIU,Zhi LIU,Shuang JIANG. Protective effect of Modified Xiao-Xian-Xiong Decoction on liver injury in rats with type 2 diabete mellitus and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 608-616.

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参考文献 28

1	中华医学会糖尿病学分会. 中国2型糖尿病防治指南（2020年版）［J］. 国际内分泌代谢杂志， 2021， 41（5）： 482-548.
2	LAI L L， LU H Q， LI W N， et al. Protective effects of quercetin and crocin in the kidneys and liver of obese Sprague-Dawley rats with type 2 diabetes： effects of quercetin and crocin on T2DM rats［J］. Hum Exp Toxicol， 2021， 40（4）： 661-672.
3	武青庭，马星雨，谢雪姣. 小陷胸汤临床应用研究近况［J］. 湖南中医杂志， 2020， 36（6）： 169-171.
4	张保国，刘庆芳. 经方小陷胸汤药理学研究与临床应用［J］. 中成药， 2012， 34（11）： 2206-2210.
5	刘冬恋，张谦，谢蕾，等. 小陷胸汤对2型糖尿病痰热互结证大鼠的影响［J］. 中成药， 2022， 44（3）： 972-976.
6	苟筱雯，赵林华，何莉莎，等. 从态靶辨证谈中医治疗2型糖尿病的用药策略［J］. 辽宁中医杂志，2020， 47（4）： 1-4.
7	王志塔，陈依楚，黄苏萍，等. 内生痰湿对2型糖尿病大鼠造模的影响［J］. 辽宁中医杂志， 2015， 42（12）： 2453-2455.
8	尤良震，于东东，方朝晖，等. 基于循证中医药学的糖尿病前期临床研究探析［J］. 中华中医药杂志， 2020， 35（3）： 1343-1346.
9	王丹玮，柏力萄，赵静，等. 中医药治疗新诊断2型糖尿病研究进展［J］. 世界科学技术-中医药现代化， 2019， 21（1）： 86-90.
10	施岚尔，聂课朝，张文婧，等. 基于网络药理学的小陷胸汤治疗2型糖尿病的药理机制［J］. 中国实验方剂学杂志， 2020， 26（4）： 198-206.
11	胡谦锋，周春祥，刘仲书，等. 中医药治疗痰湿型2型糖尿病的研究进展［J］. 辽宁中医杂志， 2016， 43（6）： 1335-1338.
12	张利民，冯德勇，邹莲霞，等. 小陷胸汤对糖尿病前期痰湿蕴热体质的影响［J］.湖南中医药大学学报， 2018， 38（4）： 467-469.
13	潘广涛，刘宇寒，周方园，等. 大黄素通过减轻氧化应激和肝细胞凋亡对脂多糖诱导急性肝损伤的保护机制研究［J］. 世界科学技术-中医药现代化， 2021， 23（7）： 2294-2301.
14	赵娟，尚文斌，于希忠. 大黄及其有效成分抗糖尿病作用机制的研究进展［J］. 时珍国医国药， 2014，25（7）： 1704-1706.
15	颜新凤，贾敏杰，冯波. 2型糖尿病肾脏病患者血清胆汁酸谱变化分析［J］. 同济大学学报（医学版）， 2022， 43（6）： 788-792.
16	ZOU W， ZHANG C， GU X， et al. Metformin in combination with malvidin prevents progression of non-alcoholic fatty liver disease via improving lipid and glucose metabolisms， and inhibiting inflammation in type 2 diabetes rats［J］. Drug Des Devel Ther， 2021， 15： 2565-2576.
17	王睿斌，卢雨征，朱静林，等. 利多卡因抑制糖尿病小鼠Kupffer细胞炎症反应及对肝脓肿形成的影响［J］. 临床肝胆病杂志， 2022， 38（6）： 1341-1346.
18	曲敬蓉，张艳艳，宿宏佳，等.黄芪多糖激活Nrf2/HO-1 信号通路改善模型大鼠糖尿病性肝损伤［J］. 中国药理学通报， 2020， 36（10）：1422-1427.
19	王巧玲，梁庆生，黄昂，等. 255例肝穿刺证实的慢性药物性肝损伤患者预后的影响因素分析［J］. 临床肝胆病杂志， 2022， 38（6）： 1334-1340.
20	邵青，索南巴吉，张滨婧，等. 2021年糖尿病相关重要临床进展回顾［J］. 中国实用内科杂志， 2022，42（5）： 391-393.
21	张昊悦，赵蓓，王业皇，等. 大黄素通过调节Nrf2/HO-1和MAPKs抑制炎症和氧化应激机制研究［J］. 中国免疫学杂志， 2021， 37（9）： 1063-1068.
22	CHEN D， ZHONG L， LI Y， et al. Changes in serum inflammatory factor interleukin-6 levels and pathology of carotid vessel walls of rats with chronic periodontitis and diabetes mellitus after the periodontal intervention［J］. Saudi J Biol Sci， 2020， 27（6）：1679-1684.
23	袁维卓，刘丽兰，张睿迪，等. 白介素-1β的分泌及其影响因素［J］.牡丹江医学院学报，2018，39（6）：86-88.
24	CHEN J， DING X， WU R， et al. Novel sesquiterpene glycoside from loquat leaf alleviates type 2 diabetes mellitus combined with nonalcoholic fatty liver disease by improving insulin resistance， oxidative stress， inflammation， and gut microbiota composition［J］. J Agric Food Chem， 2021， 69（47）： 14176-14191.
25	KLISIC A， ISAKOVIC A， KOCIC G， et al. Relationship between oxidative stress， inflammation and dyslipidemia with fatty liver index in patients with type 2 diabetes mellitus［J］. Exp Clin Endocrinol Diabetes， 2018，126（6）：371-37
26	WU Q， GAI S， ZHANG H. Asperulosidic acid， a bioactive iridoid， alleviates placental oxidative stress and inflammatory responses in gestational diabetes mellitus by suppressing NF-κB and MAPK signaling pathways［J］. Pharmacology， 2022， 107（34）： 197-205.
27	崔芳芹，胡明迹，黄丽，等. 川芎嗪通过增强糖尿病大鼠的抗氧化和抗炎作用减轻下颌下腺组织损伤［J］. 细胞与分子免疫学杂志， 2021， 37（3）： 212-218.
28	LOBODA A， DAMULEWICZ M， PYZA E， et al. Role of Nrf2/HO-1 system in development， oxidative stress response and diseases： an evolutionarily conserved mechanism［J］. Cell Mol Life Sci， 2016， 73（17）： 3221-3247.

Group	Body mass
Group	Before administration	1 weeks after administration	2 weeks after administration	3 weeks after administration		4 weeks after administration
Control	307.61±3.34	316.32±11.95	325.40±12.64	340.59±11.02	361.10±12.79
Model	303.45±22.41	265.19±30.98^*	242.24±27.48^*	234.23±24.75^*	219.89±23.82^*
Metformin	308.24±23.86	288.33±21.93	255.85±25.78	255.83±25.38	242.63±27.65
Low dose of MXD	310.42±37.47	281.80±40.95	261.11±30.92	255.01±41.42	230.78±24.81
High dose of MXD	309.96±17.18	278.04±14.79	251.36±11.24	249.07±13.67	232.56±17.48

Group	FBG level
Group	Before administration		1 weeks after administration		2 weeks after administration		3 weeks after administration		4 weeks after administration
Control	5.04±0.55	5.78±0.48		6.09±0.83		5.49±0.54		5.37±0.68
Model	18.89±1.92^*	19.24±1.56^*		18.81±1.64^*		18.72±4.04^*		17.99±2.55^*
Metformin	20.34±3.18	18.52±1.60		15.92±2.21^△△		13.55±1.79^△△		11.21±1.03^△△
Low dose of MXD	19.97±1.93	20.79±3.94		18.13±3.06		14.88±4.87^△		11.79±3.57^△△
High dose of MXD	20.34±3.20	18.66±3.07		16.04±2.81^△△		12.05±2.84^△△		11.08±2.74^△△

Group	ALT activity	AST activity
Control	83.87±19.21	110.54±16.27
Model	435.73±59.65^*	601.46±77.78^*
Metformin	166.09±31.83^△	298.42±34.49^△
Low dose of MXD	93.71±17.68^△	204.78±13.66^△
High dose of MXD	86.26±13.05^△	169.92±12.61^△

Group	IL-1β	IL-6	TNF-α
Control	23.58±6.75	31.13±4.42	72.90±12.03
Model	46.79±6.02^*	157.21±8.10^*	286.78±33.39^*
Metformin	28.49±4.26^△	84.60±5.51^△	188.02±16.60^△
Low dose of MXD	30.82±4.71^△	80.82±4.65^△	151.86±18.59^△
High dose of MXD	27.04±3.08^△	51.86±2.23^△	60.59±9.56^△

Group	SOD [λ_B/(U·mg^－1)]	GSH [m_B/(μmol·g^－1)]	MDA [m_B/(nmol·g^－1)]
Control	180.52±11.69	290.22±45.94	4.35±0.57
Model	96.85±13.76^*	38.21±5.80^*	9.77±2.16^*
Metformin	156.36±13.48^△△	55.79±7.05^△△	8.03±1.38
Low dose of MXD	129.70±16.82^△△	114.09±25.13^△△	7.35±1.31^△
High dose of MXD	145.58±15.67^△△	122.98±13.48^△△	5.09±0.86^△△