黄芩苷调节cAMP/PKA/CREB信号通路对湿疹大鼠皮肤屏障功能的影响

doi:10.11958/20230416

摘要/Abstract

摘要：

目的探讨黄芩苷（BA）调节环磷酸腺苷（cAMP）/蛋白激酶A（PKA）/cAMP反应元件结合蛋白（CREB）通路对湿疹大鼠皮肤屏障功能的影响。方法将SD大鼠随机分为对照组（NC组）、Model组、低剂量BA组（BA-L组，25 mg/kg）、中剂量BA组（BA-M组，50 mg/kg）、高剂量BA组（BA-H组，100 mg/kg）、泼尼松组（PNS组，25 mg/kg）、BA-H+cAMP抑制剂（SQ22536）组（100 mg/kg+2.13 mg/kg）、BA-H+PKA抑制剂（H-89）组（100 mg/kg+5 mg/kg），每组12只。除NC组外，其余组大鼠均构建湿疹大鼠模型。建模成功2 d后，分组进行给药处理。检测湿疹面积及严重度指数（EASI）评分、经皮肤水分流失量（TEWL）、角质层含水量（WCSC）变化；酶联免疫吸附试验（ELISA）检测大鼠血清中免疫球蛋白E（IgE）、干扰素-γ（IFN-γ）、白细胞介素-4（IL-4）水平及大鼠背部受试区皮损组织中cAMP蛋白表达；HE染色检测大鼠背部受试区皮损组织病理变化；Western blot检测大鼠背部受试区皮损组织中水通道蛋白3（AQP3）、cathelicidin相关抗菌肽（CRAMP）、p-PKA、p-CREB蛋白表达。结果与NC组比较，Model组大鼠背部受试区皮损组织病理损伤严重，EASI评分、TEWL、IgE、IL-4水平升高，WCSC、IFN-γ水平、AQP3、CRAMP、cAMP、p-PKA、p-CREB蛋白水平降低（P<0.05）。与Model组比较，BA-L组、BA-M组、BA-H组、PNS组大鼠背部受试区皮损组织病理损伤减轻，EASI评分、TEWL、IgE、IL-4水平降低，WCSC、IFN-γ水平、AQP3、CRAMP、cAMP、p-PKA、p-CREB蛋白水平升高（P<0.05）；且BA-L组、BA-M组、BA-H组上述指标变化呈剂量依赖性。SQ22536或H-89减弱了高剂量BA对湿疹大鼠皮肤屏障功能的改善作用。结论 BA可能通过激活cAMP/PKA/CREB信号通路改善湿疹大鼠皮肤屏障功能。

关键词: 湿疹, 黄芩苷, 环磷酸腺苷/蛋白激酶A/cAMP反应元件结合蛋白通路, 皮肤屏障功能

Abstract:

Objective To investigate the effect of baicalin (BA) regulating cyclic adenosine phosphate (cAMP)/protein kinase A(PKA)/cAMP response elemen-binding protein (CREB) pathway on skin barrier function in eczema rats. Methods SD rats were randomly divided into the control group (NC group), the model group, the low-dose BA group (BA-L group, 25 mg/kg), the medium-dose BA group (BA-M group, 50 mg/kg), the high-dose BA group (BA-H group, 100 mg/kg), the prednisone group (PNS group, 25 mg/kg), the BA-H+cAMP inhibitor (SQ22536) group (100 mg/kg+2.13 mg/kg) and the BA-H+PKA inhibitor (H-89) group (100 mg/kg+5 mg/kg), 12 animals in each group. Except for the NC group, eczema rat model was constructed in the other groups. Two days after successful modeling, drug administration was performed in groups. Changes of eczema area and severity index (EASI) score, transcutaneous water loss (TEWL) and cuticle water content (WCSC) were detected. Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of immunoglobulin E (IgE), interferon-γ (IFN-γ) and interleukin-4 (IL-4) in rat serum and the expression of cAMP protein in rat back lesions. HE staining was used to detect pathological changes of skin lesions on the back of rats. Western blot assay was used to detect aquaporin 3 (AQP3), cathelicidin related antimicrobial peptide (CRAMP), p-PKA, p-CREB protein expression in rat back lesions. Results Compared with the NC group, rats had serious pathological lesions on the back of the tested area, increased EASI score, TEWL, IgE and IL-4 levels, and decreased WCSC, IFN-γ, AQP3, CRAMP, cAMP, p-PKA and p-CREB protein levels in the model group (P < 0.05). Compared with the model group, pathological lesion of the tested area in the back of rats was relieved, and EASI score, TEWL, IgE and IL-4 levels were decreased, WCSC, IFN-γ levels, AQP3, CRAMP, cAMP, p-PKA and p-CREB protein were increased in the BA-L group, the BA-M group, the BA-H group and the PNS group (P<0.05). Changes of above indexes in the BA-L group, the BA-M group, the BA-H group were dose-dependent. SQ22536 or H-89 attenuated the improvement effect of high dose BA on skin barrier function in eczema rats. Conclusion BA may improve skin barrier function in eczema rats by activating cAMP/PKA/CREB signaling pathway.

Key words: eczema, baicalin, cyclic adenosine monophosphate/protein kinase A/cyclic-AMP response binding protein pathway, skin barrier function

中图分类号:

R285.5

图/表 5

参考文献 22

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组别	EASI评分/分	TEWL/ （g·m^-2·h^-1）	WCSC/%
NC组	0	8.68±0.37	63.57±3.11
Model组	7.33±0.32^a	46.52±1.89^a	18.86±0.82^a
BA-L组	6.84±0.15^b	40.14±1.57^b	25.57±1.13^b
BA-M组	6.11±0.10^bc	32.28±1.23^bc	34.43±1.66^bc
BA-H组	3.32±0.09^bcd	17.73±0.82^bcd	46.68±2.23^bcd
PNS组	3.30±0.10^bcd	17.69±0.75^bcd	46.73±2.19^bcd
BA-H+SQ22536组	6.58±0.12^e	35.52±1.63^e	30.36±1.47^e
BA-H+H-89组	6.54±0.11^e	35.48±1.57^e	30.45±1.45^e
F	3 384.000^＊	1 139.000^＊	698.800^＊

组别	EASI评分/分	TEWL/ （g·m^-2·h^-1）	WCSC/%
NC组	0	8.68±0.37	63.57±3.11
Model组	7.33±0.32^a	46.52±1.89^a	18.86±0.82^a
BA-L组	6.84±0.15^b	40.14±1.57^b	25.57±1.13^b
BA-M组	6.11±0.10^bc	32.28±1.23^bc	34.43±1.66^bc
BA-H组	3.32±0.09^bcd	17.73±0.82^bcd	46.68±2.23^bcd
PNS组	3.30±0.10^bcd	17.69±0.75^bcd	46.73±2.19^bcd
BA-H+SQ22536组	6.58±0.12^e	35.52±1.63^e	30.36±1.47^e
BA-H+H-89组	6.54±0.11^e	35.48±1.57^e	30.45±1.45^e
F	3 384.000^＊	1 139.000^＊	698.800^＊

组别	IgE/（mg/L）	IFN-γ/（ng/L）	IL-4/（ng/L）
NC组	112.28±5.44	63.32±2.79	38.67±1.73
Model组	283.36±12.25^a	24.42±1.13^a	84.45±3.83^a
BA-L组	243.43±10.20^b	33.58±1.42^b	72.29±2.97^b
BA-M组	200.12±8.45^bc	42.28±1.87^bc	61.33±2.41^bc
BA-H组	134.66±5.45^bcd	54.47±2.05^bcd	46.67±1.72^bcd
PNS组	132.28±5.61^bcd	54.36±1.98^bcd	47.72±1.66^bcd
BA-H+SQ22536组	223.37±10.54^e	35.57±1.62^e	68.87±2.69^e
BA-H+H-89组	220.69±10.31^e	35.63±1.59^e	69.03±2.58^e
F	563.800^＊	599.500^＊	445.000^＊

组别	IgE/（mg/L）	IFN-γ/（ng/L）	IL-4/（ng/L）
NC组	112.28±5.44	63.32±2.79	38.67±1.73
Model组	283.36±12.25^a	24.42±1.13^a	84.45±3.83^a
BA-L组	243.43±10.20^b	33.58±1.42^b	72.29±2.97^b
BA-M组	200.12±8.45^bc	42.28±1.87^bc	61.33±2.41^bc
BA-H组	134.66±5.45^bcd	54.47±2.05^bcd	46.67±1.72^bcd
PNS组	132.28±5.61^bcd	54.36±1.98^bcd	47.72±1.66^bcd
BA-H+SQ22536组	223.37±10.54^e	35.57±1.62^e	68.87±2.69^e
BA-H+H-89组	220.69±10.31^e	35.63±1.59^e	69.03±2.58^e
F	563.800^＊	599.500^＊	445.000^＊

组别	cAMP/（nmol/L）	AQP3/β-actin	CRAMP/β-actin	p-PKA/PKA	p-CREB/CREB
NC组	13.36±0.54	1.56±0.12	1.68±0.13	0.94±0.03	0.92±0.08
Model组	4.67±0.23^a	0.42±0.03^a	0.72±0.05^a	0.23±0.02^a	0.41±0.03^a
BA-L组	5.89±0.26^b	0.59±0.05^b	0.93±0.07^b	0.36±0.02^b	0.52±0.04^b
BA-M组	7.34±0.28^bc	0.82±0.07^bc	1.14±0.11^bc	0.49±0.05^bc	0.60±0.05^bc
BA-H组	11.32±0.43^bcd	1.28±0.11^bcd	1.36±0.12^bcd	0.82±0.07^bcd	0.84±0.08^bcd
PNS组	11.36±0.45^bcd	1.30±0.12^bcd	1.38±0.13^bcd	0.84±0.07^bcd	0.86±0.07^bcd
BA-H+SQ22536组	6.64±0.37^e	0.73±0.06^e	1.03±0.09^e	0.42±0.03^e	0.54±0.04^e
BA-H+H-89组	11.40±0.41	0.75±0.06^e	1.06±0.11^e	0.44±0.03^e	0.56±0.05^e
F	420.100^＊	135.700^＊	49.350^＊	207.300^＊	63.960^＊