黄芪建中汤靶向BMAL1调控cAMP / PKA / CREB 信号通路 对3T3-L1 细胞脂解及棕色化模型影响的研究.

Objective 　We aimed to investigate the inhibitory effect of serum containing Huangqi Jianzhong Decoction on isoproterenol (ISO) induced lipolysis and browning in 3T3-L1 adipocytes and its relationship with the regulation of the cyclic adenosine monophosphate ( cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway by brain and muscle aromatic hydrocarbon receptor nuclear transcription factor like protein-1 (BMAL1). Methods　Blank serum and Huangqi Jianzhong Decoction drug-containing serum were prepared. 3T3-L1 adipocytes were cultured in vitro and induced to differentiate and mature. They were divided into the control group, the blank group, the 2. 5% drug-containing serum group, the 5% drug-containing serum group, and the 10% drug-containing serum group. 10 mmol/ L ISO was given to all groups except for the control group to establish the hypertipolysis and browning model. Oil red O staining and enzyme-linked immunosorbent assay (ELISA) were used to detect the lipid droplet positive area ratio and the content of hydrolyzed products. Real-time PCR and Western blotting were conducted to detect the expression of lipolysis- and browning-related genes and proteins, respectively, to select the optimal volume fraction of drug-containing serum for the lentivirus transfection experiment. Mature adipocytes were transfected with si-BMAL1 to silence BMAL1. Control cells were transfected with si-NC. Then the cells were divided into the si-NC group, the containing serum si-NC group, the si-BMAL1 group, and the drug-containing serum si-BMAL1 group. After incubation with ISO, the lipid droplet positive area ratio in adipocytes was detected by oil red O staining. The contents of triglyceride (TG) and cAMP in adipocytes and free fatty acid (FFA) in medium were detected by ELISA. The protein expression levels of PKA and CREB were determined by Western blotting. The mRNA expression levels of hormone-sensitive lipase ( HSL), triglyceride lipase (ATGL), uncoupling protein 1 (UCP1), and T-box transcription factor 1 (TBX1) were determined by real-time PCR. Results　Compared with the 2. 5% and 5% drug-containing serum groups, the contents of cAMP in adipocytes and FFA in culture medium were decreased (P<0. 05), the content of TG was increased (P<0. 05), the protein expression of PKA was decreased (P<0. 05), and the mRNA expression levels of HSL and ATGL were decreased (P<0. 05) in the 10% drug-containing serum group. The relative protein expression of BMAL1 was significantly lower in 3T3-L1 adipocytes transfected with si-BMAL1 than in the si-NC group (P<0. 05), indicating that BMAL1 was successfully silenced. Compared with the si-NC group, the lipid droplet positive area ratio and TG content were decreased in the si-BMAL1 group (P<0. 05), while the FFA content in culture medium, cAMP content in adipocytes, and PKA and CREB protein expression levels were increased (P<0. 05), and the mRNA expression levels of HSL, ATGL, UCP1, and TBX1 were also increased (P<0. 05). Compared with the si-BMAL1 group, the lipid droplet positive area ratio and TG content were increased in the drug- containing serum si-BMAL1 group (P<0. 05), while the content of FFA in culture medium, the content of cAMP in adipocytes, and the protein expression levels of PKA and CREB were significantly decreased (P<0. 05) and the mRNA expression levels of HSL, ATGL, UCP1, and TBX1 were also decreased (P<0. 05). Conclusion　Drug-containing serum Huangqi Jianzhong Decoction can inhibit ISO-induced lipolysis and browning in 3T3-L1 adipocytes, the mechanism of action may be related to the regulation of the cAMP / PKA/ CREB signaling pathway by BMAL1. [ABSTRACT FROM AUTHOR]