基于PI3K/Akt/Nrf2信号通路探讨天王补心丹加减对睡眠剥夺小鼠皮肤影响的作用机制

Mechanism of Modified Tianwang Buxindan on Skin of Sleep-deprived Mice Through PI3K/Akt/Nrf2 Signaling Pathway

目的:观察天王补心丹加减(MTBD)对睡眠剥夺(SD)小鼠皮肤的作用,并探讨其作用机制。方法:将60只2月龄雌性昆明种小鼠随机分为空白组、模型组、维生素C(VC,0.08 g·kg^(-1))组和MTBD低、中、高剂量(6.5、12.5、25 g·kg^(-1))组。除空白组外,其余组均复制SD小鼠模型采用多平台水环境法行每日睡眠剥夺18 h(15:00至次日9:00),连续14 d,并从第2周起叠加腹腔注射咖啡因(CAF)7.5 mg·kg^(-1),连续7 d;造模同时,空白组、模型组灌胃生理盐水(0.01 mL·g^(-1)),其余各组均灌胃相应药物治疗。实验结束后当天进行一般表现的观察记录(如小鼠体质量、精神、毛发、皮肤等);取材后,采用苏木素-伊红(HE)和马松(Masson)染色法,光学显微镜下观察皮肤组织病理形态变化;测量皮肤厚度、皮肤含水率;生化试剂盒检测皮肤羟脯氨酸(HYP)含量、皮肤及血清超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;酶联免疫吸附测定法(ELISA)检测小鼠血清白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α和IL-1β含量;蛋白免疫印迹法检测皮肤组织Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)、磷脂酰肌醇3-激酶(PI3K)、磷酸化(p)-PI3K、蛋白激酶B(Akt)、p-Akt、核因子E2相关因子2(Nrf2)、血红素加氧酶(HO)-1、核转录因子(NF)-κB蛋白表达。结果:与空白组比较,模型组有不同程度的变化,在一般表现上,出现了诸如体质量减轻(P<0.01),精神萎靡、毛色晦暗、皮肤皱纹形成等衰老迹象;对组织标本检测,结果显示,光学显微镜下可见皮肤变薄,真表皮连接(DEJ)扁平化,胶原纤维减少;皮肤组织的厚度和含水率降低,皮肤组织HYP含量显著减少(P<0.01),皮肤和血清SOD活性显著下降(P<0.01)及MDA含量显著增加(P<0.01);血清IL-6、TNF-α、IL-1β含量显著增加(P<0.01);皮肤ColⅠ、ColⅢ、p-PI3K/PI3K、p-Akt/Akt、Nrf2及HO-1蛋白表达明显减少(P<0.05,P<0.01),NF-κB表达增加(P<0.01)。与模型组比较,VC组和MTBD低剂量组皮肤的含水率、HYP含量、SOD活性及ColⅠ、ColⅢ、p-PI3K/PI3K蛋白表达增加(P<0.05,P<0.01),血清MDA含量减少(P<0.05),此外MTBD低剂量组还检测出血清IL-6、IL-1β含量减少(P<0.05);而MTBD中、高剂量组的上述各指标均有改善(P<0.05,P<0.01)。结论:SD会加促SD模型鼠皮肤老化进程。MTBD可以改善这种现象,发挥抗炎、抗氧化作用,其作用机制可能与激活PI3K/Akt/Nrf2信号通路相关。

Objective: To observe the effect of modified Tianwang Buxindan(MTBD) on the skin of sleep-deprived(SD) mice and investigate its mechanism. Method: Sixty 2-month-old female Kunming mice were randomly divided into a blank group, a model group, a vitamin C(VC, 0.08 g·kg^(-1)), and MTBD low-, medium-, and high-dose groups(6.5, 12.5, 25 g·kg^(-1)). Except for the blank group, the other groups were subjected to SD mouse model induction(using multiple platform water environment method for 18 hours of sleep deprivation daily from 15:00 to next day 9:00), continuously for 14 days, and caffeine(CAF, 7.5 mg·kg^(-1)) was injected intraperitoneally from the 2nd week onwards, continuously for 7 days. While modeling, the blank group and the model group were administered with normal saline(0.01 mL·g^(-1)), and the other groups received corresponding drugs for treatment. On the day of the experiment, general observations were recorded(such as body weight, spirit, fur, and skin). After sampling, skin tissue pathological changes were observed under an optical microscope using hematoxylin-eosin(HE) and Masson staining methods. Skin thickness and skin moisture content were measured. Biochemical assay kits were used to detect skin hydroxyproline(HYP) content, skin and serum superoxide dismutase(SOD) activity, and malondialdehyde(MDA) content. Enzyme-linked immunosorbent assay(ELISA) was used to detect serum interleukin(IL)-6, tumor necrosis factor(TNF)-α, and IL-1β levels in mice. Western blot was used to detect skin tissue type Ⅰcollagen(ColⅠ), type Ⅲ collagen(ColⅢ), phosphatidylinositol 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), p-Akt, nuclear factor E_(2)-related factor 2(Nrf2), heme oxygenase(HO)-1, and nuclear factor(NF)-κB protein expression. Result: Compared with the blank group, the model group showed varying degrees of changes. In general, signs of aging such as reduced body weight(P<0.01), listlessness, dull fur color, and formation of wrinkles on the skin appeared. Tissue specimen testing revealed skin thinning, flattening of the dermoepidermal junction(DEJ), and reduced collagen fibers under the optical microscope. Skin thickness and moisture content decreased, skin tissue HYP content significantly decreased(P<0.01), skin and serum SOD activity significantly decreased(P<0.01), and MDA content significantly increased(P<0.01). Serum IL-6, TNF-α, and IL-1β levels significantly increased(P<0.01). Skin Col Ⅰ, Col Ⅲ, p-PI3K/PI3K, p-Akt/Akt, Nrf2, and HO-1 protein expression significantly decreased(P<0.05, P<0.01), and NF-κB expression increased(P<0.01). Compared with the model group, the VC group and the MTBD low-dose group showed increased skin moisture content, HYP content, SOD activity, and Col Ⅰ, Col Ⅲ, p-PI3K/PI3K protein expression(P<0.05, P<0.01), and decreased serum MDA content(P<0.05). In addition, a decrease in serum IL-6 and IL-1β levels was detected in the MTBD low-dose group(P<0.05), while the above indicators in the MTBD medium-and high-dose groups improved(P<0.05, P<0.01). Conclusion: Sleep deprivation accelerates the aging process of the skin in SD model mice. MTBD can improve this phenomenon, exerting anti-inflammatory and antioxidant effects, and its mechanism of action may be related to the activation of the PI3K/Akt/Nrf2 signaling pathway.