Song Fei, Wang Lihua, Mu Jing, Ma Huisheng
School of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan, Ningxia 750004, China.
Key Laboratory of Ningxia Minority Medicine Modernization Ministry of Education, Yinchuan, Ningxia 750003, China.
Evid Based Complement Alternat Med. 2022 Jun 28;2022:4156330. doi: 10.1155/2022/4156330. eCollection 2022.
To investigate the effects and corresponding mechanisms of total flavonoids (TFL) from Lycium barbarum leaves on photoaged human dermal fibroblasts (HDFs).
Crude TFL was extracted with 70% ethanol, and a Rutin standard curve was drawn using the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry method to calculate its yield and mass concentration. After that, the photoaging HDFs model was established by UVA combined with 8-MOP. CCK-8 was performed to assess the influence of TFL on the proliferation of HDFs and photoaging HDFs. -galactosidase (SA--gal) staining and activity assays were performed to evaluate the activity of SA--gal and the rate of SA--gal-positive cells in HDFs cells. The level of skin ECM proteins and oxidative stress-related substances in HDFs cells of each group was determined by ELISA and biochemical detection, respectively. Apoptosis of HDFs in each group was assessed by flow cytometry. The expressions of MAPK signaling pathway-related proteins in HDFs were detected by western blot.
The yield rate of TFL extracted by 70% ethanol was 41.9%, and its purity rate was 34.6%. TFL at 25, 50, and 100 g/mL was able to greatly promote the proliferation of HDFs. A photoaged HDFs model was successfully constructed by combining UVA irradiation at 9 J/cm and 8-MOP at 50 mg/L. TFL treatment could significantly inhibit apoptosis, SA--gal-positive cell staining rate, SA--gal activity, lactate dehydrogenase (LDH) leakage, and malondialdehyde (MDA) content in photoaged HDFs. Further, TFL increased the proliferative activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, type I collagen (Col I), hydroxyproline (HYP), and hyaluronic acid (HA) level of photoaged HDFs in a dose-dependent manner. Additional experiments suggested that TFL played a protective role by downregulating MAPK signaling pathway activity in photoaged HDFs cells.
TFL could inhibit oxidative stress and apoptosis, promote cell proliferation and the level of ECM-related component proteins, and participate in antiphotoaging in a concentration-dependent manner. The protective role of TFL in photoaged HDFs might be related to its inhibition of MAPK signaling pathways.
探讨枸杞叶总黄酮（TFL）对光老化人皮肤成纤维细胞（HDFs）的作用及其相应机制。
用70%乙醇提取TFL粗品，采用亚硝酸钠-硝酸铝-氢氧化钠比色法绘制芦丁标准曲线，计算其得率和质量浓度。之后，通过紫外线A（UVA）联合8-甲氧基补骨脂素（8-MOP）建立光老化HDFs模型。采用细胞计数试剂盒-8（CCK-8）法评估TFL对HDFs及光老化HDFs增殖的影响。进行β-半乳糖苷酶（SA-β-gal）染色及活性检测，以评估HDFs细胞中SA-β-gal的活性及SA-β-gal阳性细胞率。分别采用酶联免疫吸附测定（ELISA）法和生化检测法测定各组HDFs细胞中皮肤细胞外基质（ECM）蛋白水平及氧化应激相关物质水平。采用流式细胞术评估各组HDFs的凋亡情况。通过蛋白质免疫印迹法检测HDFs中丝裂原活化蛋白激酶（MAPK）信号通路相关蛋白的表达。
70%乙醇提取的TFL得率为41.9%，纯度为34.6%。25、50和100μg/mL的TFL能够显著促进HDFs的增殖。通过9J/cm的UVA照射与50mg/L的8-MOP联合，成功构建了光老化HDFs模型。TFL处理可显著抑制光老化HDFs的凋亡、SA-β-gal阳性细胞染色率、SA-β-gal活性、乳酸脱氢酶（LDH）泄漏及丙二醛（MDA）含量。此外，TFL以剂量依赖性方式提高光老化HDFs的增殖活性、超氧化物歧化酶（SOD）活性、过氧化氢酶（CAT）活性、I型胶原蛋白（Col I）、羟脯氨酸（HYP）和透明质酸（HA）水平。进一步实验表明，TFL通过下调光老化HDFs细胞中MAPK信号通路活性发挥保护作用。
TFL可抑制氧化应激和细胞凋亡，促进细胞增殖及ECM相关成分蛋白水平，且呈浓度依赖性参与抗光老化作用。TFL对光老化HDFs的保护作用可能与其对MAPK信号通路的抑制有关。
