抗菌肽LL-37对糖尿病心肌病大鼠心肌损伤的保护作用及其机制

doi:10.13481/j.1671-587X.20220217

摘要/Abstract

摘要： 目的

探讨抗菌肽LL-37对糖尿病心肌病（DCM）大鼠心肌损伤的影响，并阐明其可能的作用机制。

方法

将48只SD大鼠随机分为正常对照组、模型组、低剂量LL-37组（0.5 mg·kg^－1）和高剂量LL-37组（2.0 mg·kg^－1），每组12只。除正常对照组外，其他各组大鼠均通过高脂饲料喂养联合腹腔注射链脲佐菌素（STZ）构建DCM模型。模型构建成功后，低和高剂量LL-37组大鼠给予抗菌肽LL-37干预，每天1次，连续6周，而正常对照组和模型组大鼠腹腔注射等体积生理盐水。监测各组大鼠体质量和空腹血糖（FBG）水平，检测各组大鼠血清总胆固醇（TC）和甘油三酯（TG）水平，心脏超声检测各组大鼠心功能，HE染色观察各组大鼠心肌组织病理形态表现，TUNEL染色检测各组大鼠心肌细胞凋亡率，ELISA法检测各组大鼠心肌组织中炎症因子白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）水平，Western blotting法检测各组大鼠心肌组织中Toll样受体4（TLR4）、核因子κB p65（NF-κB p65）和NF-κB抑制因子α（IκBα）等蛋白表达水平。

结果

与正常对照组比较，模型组大鼠心肌损伤严重，左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）和体质量明显降低（P<0.05），左心室舒张末期内径（LVEDd）、左心室收缩末期内径（LVEDs）、FBG、TC、TG和心肌细胞凋亡率明显升高（P<0.05），心肌组织中IL-1β、IL-6和TNF-α水平及TLR4、NF-κB p65蛋白表达水平均明显升高（P<0.05），而IκBα蛋白表达水平明显降低（P<0.05）；与模型组和低剂量LL-37组比较，高剂量LL-37组大鼠心肌损伤明显改善，LVEF、LVFS和体质量明显升高（P<0.05），LVEDd、LVEDs、FBG、TC、TG和心肌细胞凋亡率明显降低（P<0.05），心肌组织中IL-1β、IL-6和TNF-α水平及TLR4、NF-κB p65蛋白表达水平均明显降低（P<0.05），IκBα蛋白表达水平明显升高（P<0.05）；与模型组比较，低剂量LL-37组大鼠以上各指标差异无统计学意义（P>0.05）。

结论

抗菌肽LL-37可改善DCM大鼠心功能，抑制心肌组织炎症和细胞凋亡，对DCM大鼠心肌损伤具有一定保护作用，并能抑制TLR4/NF-κB信号通路。

关键词: 糖尿病心肌病, 心肌损伤, 抗菌肽LL-37, Toll样受体4, 核因子κB

Abstract: Objective

To investigate the effect of antibacterial peptide LL-37 on the myocardial injury in the diabetic cardiomyopathy （DCM） rats， and to elucidate its possible mechanism.

Methods

A total of 48 SD rats were randomly divided into normal control group， model group， low dose of LL-37 group （0.5 mg·kg^－1） and high dose of LL-37 group（2.0 mg·kg^－1），with 12 rats in each group. Except for normal control group， the rats in other groups were fed with high-fat diet combined with intraperitoneal injection of streptozotocin （STZ） to establish the DCM models. After the models were successfully established， the LL-37 drug intervention was performed in low and high doses of LL-37 groups， once a day for 6 weeks. The body weights and fasting blood glucose （FBG） levels of the rats in various groups were monitored； the serum total cholesterol （TC） and triglyceride （TG） levels of the rats in various groups were detected. Cardiac ultrasonography was used to detect the cardiac function of the rats.The pathomorphology of myocardium tissue of the rats in various groups were observed by HE staining. TUNEL staining was used to detect the apoptotic rates of myocardial cells of the rats in various groups. The levels of inflammatory cytokines interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in myocardium tissue of the rats in various groups were detected by ELISA. The expression levels of Toll-like receptor 4 （TLR4）， nuclear factor kappa B p65 （NF-κB p65） and NF-κB inhibitor α（IκBα） proteins in myocardium tissue of the rats in various groups were detected by Western blotting method.

Results

Compared with normal control group， the myocardial injury of the rats in model group was serious，the left ventricular ejection fraction （LVEF），the left ventricular fractional shortening（LVFS） and the body weight were significantly decreased （P<0.05），the left ventricular end diastolic diameter （LVEDd），the left ventricular end systolic diameter （LVEDs），the levels of FBG， TC， TG and the apoptotic rate of myocardial cells were significantly increased （P<0.05），the levels of IL-1β， IL-6 and TNF-α and the expression levels of TLR4 and NF-κB p65 proteins in myocardium tissue of the rats were significantly increased （P<0.05）， while the expression level of IκBα protein was significantly decreased （P<0.05）. Compared with model group and low dose of LL-37 group， the myocardial injury of the rats in high dose of LL-37 group was significantly improved，the LVEF， LVFS and the body weight were significantly increased （P<0.05）， while the LVEDd， LVEDs， the levels of FBG， TC， TG and the apoptotic rates of myocardial cells were significantly decreased （P<0.05）， the levels of IL-1β， IL-6 and TNF-α and the expression levels of TLR4 and NF-κB p65 proteins in myocardium tissue were significantly decreased （P<0.05）， while the expression level of IκBα protein was significantly increased （P<0.05）. Compared with model group， there were no significant changes in all above indexes in low dose of LL-37 group （P>0.05）.

Conclusion

Antimicrobial peptide LL-37 can improve the cardiac function of DCM rats， inhibit the inflammation of myocardium tissue and apoptosis， protect the myocardial injury， and inhibit the activation of TLR4/NF-κB signaling pathway.

Key words: Diabetic cardiomyopathy, Myocardial injury, Antibacterial peptide LL-37, Toll-like receptor 4, Nuclear factor-kappa B

中图分类号:

R285.5

武春艳,汪剑英,曾海燕,谭少文. 抗菌肽LL-37对糖尿病心肌病大鼠心肌损伤的保护作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(2): 399-406.

Chunyan WU,Jianying WANG,Haiyan ZENG,Shaowen TAN. Protective effect of antimicrobial peptide LL-37 on myocardial injury in rats with diabetic cardiomyopathy and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 399-406.

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Group	Body weight
Group	Before intervention	2 weeks after intervention	4 weeks after intervention	6 weeks after intervention
Control	349.32±6.35	387.13±14.04^○	401.61±12.13^○	438.26±13.46^○
Model	295.07±13.05^*	224.27±18.31^*○	209.38±17.33^*○	140.18±12.88^*○
Low dose of LL-37	290.34±20.90	227.03±10.52^○	218.65±10.90^○	147.05±20.60^○
High dose of LL-37	296.84±3.96^△#	274.18±12.24^△#○	251.92±27.40^△#○	248.16±20.41^△#○

Group	FBG level
Group	Before intervention	2 weeks after intervention	4 weeks after intervention	6 weeks after intervention
Control	5.12±0.61	5.26±0.75	5.60±0.80	5.72±0.10
Model	21.00±1.42^*	24.73±1.89^*○	26.10±1.85^*○	28.86±2.55^*○
Low dose of LL-37	21.40±0.66	23.83±1.23^○	25.01±2.73^○	27.36±2.74^○
High dose of LL-37	22.34±1.47^△#	20.12±1.61^△#○	17.44±1.50^△#○	14.50±2.39^△#○

Group	TC	TG
Control	1.38±0.18	0.84±0.20
Model	2.71±0.20^*	7.95±0.44^*
Low dose of LL-37	2.67±0.34	7.66±0.85
High dose of LL-37	2.02±0.19^△#	4.31±0.64^△#

Group	LVEDd (l/mm)	LVEDs (l/mm)	LVEF (η/%)	LVFS (η/%)
Control	2.81±0.20	1.27±0.16	81.30±5.71	45.37±3.09
Model	3.81±0.10^*	2.82±0.18^*	52.40±4.16^*	32.90±2.30^*
Low dose of LL-37	3.74±0.27	2.57±0.53	53.87±3.86	34.41±1.33
High dose of LL-37	2.74±0.11^△#	1.41±0.10^△#	76.39±4.15^△#	44.18±2.79^△#

Group	IL-1β	IL-6	TNF-α
Control	10.47±1.65	18.71±3.64	130.41±15.84
Model	20.44±2.87^*	82.66±7.33^*	286.71±21.14^*
Low dose of LL-37	19.64±2.64	79.84±1.55	280.51±10.33
High dose of LL-37	13.16±1.53^△#	33.67±1.98^△#	186.65±16.05^△#