School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515, China; Department of Traditional Chinese Medicine, People's Hospital of Yangjiang, Yangjiang, 529500, China.
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515, China.
J Ethnopharmacol. 2019 Aug 10;240:111937. doi: 10.1016/j.jep.2019.111937. Epub 2019 May 7.
Atopic dermatitis (AD), a disorder prevalent during childhood and adulthood, seriously affects the patient's quality of life. Although Huang-Lian-Jie-Du-Tang (HLJDT) has shown anti-inflammatory effects in previous studies, its effects and mechanism of action underlying AD disorder are still largely unknown.
This study explored the anti-inflammatory and immunomodulatory effects of HLJDT on the AD-like dermal disorder, induced in vitro by lipopolysaccharide (LPS)-triggered inflammation, and in vivo by 2,4-dinitrochlorobenzene (DNCB).
In vivo HLJDT effects were investigated by determining the severity of dermatitis, which consisted of observing signs of skin lesions, visually and through haematoxylin and eosin (HE) staining, in mouse ears and dorsal skin, measuring serum levels of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, interferon (IFN)-γ, the tumour necrosis factor (TNF)-α, and determining the splenic index, number of splenic CD4/CD8 T-lymphocytes, as well as the phosphorylation levels of mitogen-activated protein kinases (including MAPKs-p38, ERK, and JNK), IκB-α, and nuclear factor kappa B (NF-κB) (p65) within dermal lesions. Morphological changes in LPS-induced inflammation were observed under a microscope, and ELISA and qPCR assays were used to measure IL-1α, IL-1β, IL-6, and TNF-α expression levels. The protein expression levels of P-ERK/ERK, P-p38/p38, P-JNK/JNK, P-IKβ-α, and P-p65 were measured through western blotting. Additionally, p65 expression was assessed by immunofluorescence, and LPS binding to RAW264.7 cell membrane was studied with laser confocal microscopy.
HLJDT could remarkably mitigate DNCB-induced AD-like lesion symptoms, alleviating inflammatory mediator infiltration in mouse ears and dorsal skin tissue, down-regulating serum expression levels of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IFN-γ, and TNF-α, normalising the splenic CD4/CD8 T-lymphocyte ratio, and inactivating MAPKs (including p38, ERK, and JNK), IκB-α, and NF-κB (p65) in dorsal skin. Furthermore, HLJDT inhibited LPS-induced differentiation of RAW264.7 cells, as evidenced by the decreased protein and mRNA expression of IL-1α, IL-1β, IL-6, and TNF-α. Additionally, it decreased ERK, p38, JNK, IKβ-α, and p65 phosphorylation levels in the MAPKs/NF-κB pathway, inhibited p65 nuclear translocation, and reduced LPS binding to the RAW264.7 cell membrane.
HLJDT significantly improved AD-like symptoms via inhibition of the MAPKs/NF-κB pathway. Therefore, administration of HLJDT might be a potential treatment for AD in the clinical setting.
特应性皮炎（AD）是一种在儿童和成年期普遍存在的疾病，严重影响患者的生活质量。虽然黄连解毒汤（HLJDT）在先前的研究中显示出抗炎作用，但它对 AD 疾病的作用机制仍在很大程度上未知。
本研究旨在探讨 HLJDT 对脂多糖（LPS）触发的炎症诱导的体外 AD 样皮肤疾病和 2,4-二硝基氯苯（DNCB）诱导的体内 AD 样皮肤疾病的抗炎和免疫调节作用。
通过观察小鼠耳朵和背部皮肤的皮肤损伤迹象、通过苏木精和伊红（HE）染色进行肉眼观察、测量血清中白细胞介素（IL）-1α、IL-1β、IL-2、IL-4、IL-5、IL-6、干扰素（IFN）-γ、肿瘤坏死因子（TNF）-α水平，以及测量脾指数、脾中 CD4/CD8 T 淋巴细胞数量、真皮病变中丝裂原激活蛋白激酶（包括 MAPKs-p38、ERK 和 JNK）、IκB-α 和核因子 kappa B（NF-κB）（p65）的磷酸化水平，来研究体内 HLJDT 的作用。通过显微镜观察 LPS 诱导的炎症的形态变化，使用 ELISA 和 qPCR 测定 IL-1α、IL-1β、IL-6 和 TNF-α的表达水平。通过 Western blot 测定 P-ERK/ERK、P-p38/p38、P-JNK/JNK、P-IKβ-α 和 P-p65 的蛋白表达水平。通过免疫荧光测定 p65 表达，通过激光共聚焦显微镜研究 LPS 与 RAW264.7 细胞膜的结合。
HLJDT 可显著减轻 DNCB 诱导的 AD 样病变症状，减轻小鼠耳朵和背部皮肤组织中炎症介质的浸润，下调血清中 IL-1α、IL-1β、IL-2、IL-4、IL-5、IL-6、IFN-γ和 TNF-α的表达水平，使脾中 CD4/CD8 T 淋巴细胞比值正常化，并使背部皮肤中的 MAPKs（包括 p38、ERK 和 JNK）、IκB-α 和 NF-κB（p65）失活。此外，HLJDT 抑制 LPS 诱导的 RAW264.7 细胞分化，这表现在 IL-1α、IL-1β、IL-6 和 TNF-α的蛋白和 mRNA 表达降低。此外，它降低了 MAPKs/NF-κB 通路中 ERK、p38、JNK、IκB-α 和 p65 的磷酸化水平，抑制了 p65 的核转位，并减少了 LPS 与 RAW264.7 细胞膜的结合。
HLJDT 通过抑制 MAPKs/NF-κB 通路显著改善 AD 样症状。因此，HLJDT 的给药可能是 AD 临床治疗的一种潜在方法。
