Department of Biological Sciences, School of Medical and Life Sciences, Sunway University, No.5 Jalan Universiti, Bandar Sunway, 47500, Subang Jaya, Selangor Darul Ehsan, Malaysia.
Research Centre for Nano-materials and Energy Technology (RCNMET), School of Engineering and Technology, Sunway University, No.5 Jalan Universiti, Bandar Sunway, 47500, Subang Jaya, Selangor Darul Ehsan, Malaysia.
J Ethnopharmacol. 2022 Dec 5;299:115621. doi: 10.1016/j.jep.2022.115621. Epub 2022 Aug 17.
Lignosus rhinocerus, also known as Tiger Milk Mushroom has been used traditionally to treat a variety of human conditions, including asthma, diabetes, respiratory disease, skin allergy, and food poisoning. The reported activities of Lignosus rhinocerus extracts include anti-inflammatory, anti-oxidant, anti-asthmatic, anti-microbial, anti-cancer, neuroprotection, and immune modulation effects. However, its effect on human skin is not well documented, including human skin exposed to ultraviolet light (UV). Exposure to UV can trigger various cellular responses, including inflammation, oxidative stress, DNA damage, cell death, and cellular aging.
The study aims to investigate the effects of methanolic extract prepared from cultured Lignosus rhinocerus (herein referred to as TM02 and its methanol extract as TM02-ME) on UV-irradiated human keratinocytes.
Powdered stock of TM02 was dissolved and sequentially extracted with different solvents to prepare the extracts and the methanol extract was subsequently characterized based on its bio-activities on HaCaT human keratinocytes. The keratinocytes were pre-treated with the methanol extract followed by UV-irradiation. Cellular responses of the HaCaT cells such as cell viability, DNA damage, as well as gene and protein expressions that were responsive to the treatments, were characterized by using bio-assays, including reverse-transcription based PCR, Western blot, cell viability, and mitochondrial Cytochrome C release assays.
TM02-ME protected HaCaT cells from UV-induced DNA damage and cell death in a dose-dependent manner. Pre-treatment of HaCaT cells with TM02-ME led to a 39% reduction of cyclobutane pyrimidine dimers (CPD) and up-regulated the gene expression of REV1 and SPINK5 in UVB-irradiated HaCaT cells when compared to the control. In addition, TM-02-ME treated HaCaT cells increased the expression of BCL-XL and BCL-2 proteins which coincided with the down-regulation of mitochondrial Cyt. C release in the UV-B irradiated HaCaT cells. The results were further supported by data that showed the stable clones of HaCaT cells stably expressed BCL-XL were resistant to UVB-induced cell death.
The results showed that TM02-ME confers photoprotective activities to UVB-irradiated HaCaT cells, leading to a reduction in DNA damage and cell death as well as up-regulated the expression of REV1 and SPINK5 which are involved in DNA repair and skin barrier function, respectively. The up-regulation of pro-survival members of the BCL-2 family by TM02-ME confers protection against UVB-induced cell death.
ETHNOPHARMACOLOGICAL 相关性：Lignosus rhinocerus，也被称为虎乳灵芝，传统上被用于治疗各种人类疾病，包括哮喘、糖尿病、呼吸道疾病、皮肤过敏和食物中毒。Lignosus rhinocerus 提取物的报道活动包括抗炎、抗氧化、抗哮喘、抗微生物、抗癌、神经保护和免疫调节作用。然而，它对人类皮肤的影响尚未得到很好的记录，包括人类皮肤暴露在紫外线下（UV）。暴露于 UV 会引发各种细胞反应，包括炎症、氧化应激、DNA 损伤、细胞死亡和细胞衰老。
本研究旨在探讨从培养的 Lignosus rhinocerus（以下简称 TM02）制备的甲醇提取物（简称 TM02-ME）对 UV 照射的人角质形成细胞的影响。
将 TM02 的粉末状库存溶解并依次用不同溶剂提取以制备提取物，并根据其对 HaCaT 人角质形成细胞的生物活性对甲醇提取物进行特征描述。在用甲醇提取物预处理后，角质形成细胞接受 UV 照射。通过生物测定法，包括基于逆转录的 PCR、Western blot、细胞活力和线粒体细胞色素 C 释放测定法，对 HaCaT 细胞的细胞反应，如细胞活力、DNA 损伤以及对处理有反应的基因和蛋白质表达进行特征描述。
TM02-ME 以剂量依赖的方式保护 HaCaT 细胞免受 UV 诱导的 DNA 损伤和细胞死亡。与对照相比，用 TM02-ME 预处理 HaCaT 细胞可使细胞中环丁烷嘧啶二聚体（CPD）减少 39%，并上调 UVB 照射的 HaCaT 细胞中 REV1 和 SPINK5 的基因表达。此外，TM-02-ME 处理的 HaCaT 细胞增加了 BCL-XL 和 BCL-2 蛋白的表达，同时伴随着线粒体 Cyt. C 在 UVB 照射的 HaCaT 细胞中的释放下调。这一结果得到了进一步支持，数据表明，稳定表达 BCL-XL 的 HaCaT 细胞的稳定克隆对 UVB 诱导的细胞死亡具有抗性。
结果表明，TM02-ME 赋予 UVB 照射的 HaCaT 细胞光保护活性，导致 DNA 损伤和细胞死亡减少，以及上调参与 DNA 修复和皮肤屏障功能的 REV1 和 SPINK5 的表达。TM02-ME 上调 BCL-2 家族的促生存成员赋予对 UVB 诱导的细胞死亡的保护。
