DataSheet6_The Chinese herbal medicine Dai-Zong-Fang promotes browning of white adipocytes in vivo and in vitro by activating PKA pathway to ameliorate obesity.ZIP

Introduction: The global prevalence of obesity is rising rapidly. Conversion of white adipose tissue (WAT) into beige adipose tissue with heat-consuming characteristics, i.e., WAT browning, effectively inhibits obesity. Dai-Zong-Fang (DZF), a traditional Chinese medicine formula, has long been used to treat metabolic syndrome and obesity. This study aimed to explore the pharmacological mechanism of DZF against obesity.
Methods:In vivo, C57BL/6J mice were fed high-fat diets to establish the diet-induced obese (DIO) model. DZF (0.40 g/kg and 0.20 g/kg) and metformin (0.15 g/kg, positive control drug) were used as intervention drugs for six weeks, respectively. The effects of DZF on body size, blood glucose and lipid level, structure and morphology of adipocytes and browning of inguinal WAT (iWAT) in DIO mice were observed. In vitro, mature 3T3-L1 adipocytes were used as the model. Concentrations of DZF (0.8 mg/mL and 0.4 mg/mL) were selected according to the Cell Counting Kit-8 (CCK8). After 2d intervention, lipid droplet morphology was observed by BODIPY493/503 staining, and mitochondria number was observed by mito-tracker Green staining. H-89 dihydrochloride, a PKA inhibitor, was used to observe the change in browning markers′ expression. The expression levels of browning markers UCP1 and PGC-1α and key molecules of PKA pathway were detected in vivo and in vitro.
Results:In vivo, compared with vehicle control group, 0.40 g/kg DZF significantly reduced obesity in DIO mice from body weight, abdomen circumference, Lee′s index, and WAT/body weight (p < 0.01 or p < 0.001). 0.40 g/kg DZF also significantly reduced fasting blood glucose (FBG), serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) (p < 0.01 or p < 0.001). The iWAT′s morphology and mitochondria were browning after DZF intervention. In HE-staining, the lipid droplets became smaller, and the number of mitochondria increased. The mitochondrial structure was remodeled under the electron microscope. The expression of UCP1, PGC-1α and PKA was elevated in iWAT detected by RT-qPCR (p < 0.05 or p < 0.001). In vitro, compared with the control group, 0.8 mg/mL DZF intervention significantly increased the number of mitochondria and expression of UCP1, PGC-1α, PKA, and pCREB (p < 0.05 or p < 0.01). In contrast, UCP1 and PGC-1α expression were significantly reversed after adding PKA inhibitor H-89 dihydrochloride.
Conclusion: DZF can promote UCP1 expression by activating the PKA pathway, thereby promoting browning of WAT, attenuating obesity, and reducing obesity-related glucose and lipid metabolism abnormalities, indicating that DZF has the potential to be selected as an anti-obesity drug to benefit obese patients.

引言：全球肥胖患病率正快速攀升。将具有产热特性的白色脂肪组织（white adipose tissue, WAT）转化为米色脂肪组织，即白色脂肪组织褐变（WAT browning），可有效抑制肥胖。代赭石复方（Dai-Zong-Fang, DZF）是一种传统中药方剂，长期以来被用于治疗代谢综合征与肥胖。本研究旨在探究DZF对抗肥胖的药理学机制。
方法：体内实验中，选用C57BL/6J小鼠，通过高脂饮食构建饮食诱导肥胖（diet-induced obese, DIO）模型。分别以0.40 g/kg、0.20 g/kg剂量的DZF，以及0.15 g/kg的二甲双胍（阳性对照药物）作为干预药物，干预时长为6周。观察DZF对饮食诱导肥胖小鼠的体况、血糖血脂水平、脂肪细胞结构与形态，以及腹股沟白色脂肪组织（inguinal WAT, iWAT）褐变的影响。体外实验中，以成熟3T3-L1脂肪细胞作为模型。依据细胞计数试剂盒-8（Cell Counting Kit-8, CCK8）的检测结果，选取0.8 mg/mL、0.4 mg/mL两个浓度的DZF进行干预。干预2天后，采用BODIPY493/503染色观察脂滴形态，采用线粒体追踪绿色染色（mito-tracker Green staining）观察线粒体数量。使用蛋白激酶A（PKA）抑制剂H-89二盐酸盐，观察褐变标志物表达的变化。分别在体内与体外实验中，检测褐变标志物解偶联蛋白1（UCP1）、过氧化物酶体增殖物激活受体γ辅激活因子1α（PGC-1α），以及蛋白激酶A通路关键分子的表达水平。
结果：体内实验方面，与溶剂对照组相比，0.40 g/kg剂量的DZF可显著降低饮食诱导肥胖小鼠的体重、腹围、Lee氏指数以及白色脂肪组织/体重比值（p < 0.01 或 p < 0.001）。0.40 g/kg剂量的DZF还可显著降低空腹血糖（fasting blood glucose, FBG）、血清甘油三酯（serum triglycerides, TG）、总胆固醇（total cholesterol, TC）以及低密度脂蛋白胆固醇（low-density lipoprotein cholesterol, LDL-C）水平（p < 0.01 或 p < 0.001）。经DZF干预后，腹股沟白色脂肪组织出现褐变，形态与线粒体状态发生改变：HE染色显示脂滴变小，线粒体数量增加；电镜下可见线粒体结构发生重塑。实时定量聚合酶链反应（RT-qPCR）检测结果显示，腹股沟白色脂肪组织中UCP1、PGC-1α以及PKA的表达水平升高（p < 0.05 或 p < 0.001）。体外实验方面，与对照组相比，0.8 mg/mL剂量的DZF干预可显著增加线粒体数量，并提升UCP1、PGC-1α、PKA以及pCREB的表达水平（p < 0.05 或 p < 0.01）。与之相反，加入PKA抑制剂H-89二盐酸盐后，UCP1与PGC-1α的表达水平被显著逆转。
结论：DZF可通过激活蛋白激酶A通路促进UCP1的表达，进而推动白色脂肪组织褐变，缓解肥胖并改善肥胖相关的糖脂代谢异常，表明DZF具备开发为抗肥胖药物以惠及肥胖患者的潜力。