Mechanism of Qingrun Prescription-containing Serum Improving Insulin Resistance in HepG2 Cells via Branched-chain α-keto Acid Dehydrogenase Regulation of Branched-chain Amino Acids （BCAAs）/mTOR Pathway
10.13422/j.cnki.syfjx.20250201
- VernacularTitle:清润方含药血清通过支链α-酮酸脱氢酶调控BCAAs/mTOR信号通路改善HepG2细胞胰岛素抵抗的作用机制探讨
- Author:
Xiangwei BU
1
;
Xiaohui HAO
1
;
Runyun ZHANG
1
;
Meizhen ZHANG
2
;
Ze WANG
1
;
Haoshuo WANG
1
;
Jie WANG
3
;
Qing NI
1
;
Lan LIN
1
Author Information
1. Guang'anmen Hospital，China Academy of Chinese Medicine Sciences，Beijing 100053，China
2. First Teaching Hospital of Tianjin University of Traditional Chinese Medicine（TCM），Tianjin 300381，China
3. Shunyi Hospital，Beijing TCM Hospital，Beijing 101300，China
- Publication Type:Journal Article
- Keywords:
Qingrun prescription;
type 2 diabetes mellitus;
insulin resistance;
metabolic reprogramming;
branched-chain amino acids
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2025;31(9):90-98
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo investigate the effect of Qingrun prescription（QRP）-containing serum on improving insulin resistance in HepG2 cells and its potential mechanisms. MethodsAn insulin resistance model was established in HepG2 cells with 1×10-6 mol·L-1 insulin. Branched-chain α-keto acid dehydrogenase （BCKDH） gene silencing was achieved using siRNA， and the cells were divided into 8 groups： normal group， model group （1×10-6 mol·L-1 insulin）， metformin group （1 mmol·L-1 metformin）， high-， medium-， and low-dose QRP groups （20%， 10%， and 5% QRP-containing serum， respectively）， QRP + siRNA-silenced BCKDH （si-BCKDH） group （10% QRP-containing serum + si-BCKDH）， and QRP + si-NC group （10% QRP-containing serum + si-NC）. Glucose levels in the supernatant were measured with a glucose assay kit， while glycogen content was assessed using a glycogen assay kit. Levels of branched-chain amino acids （BCAAs） and branched-chain keto acids （BCKAs） were determined using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. mRNA transcription and protein expression levels of BCKDH， dishevelled， Egl-10， and pleckstrin （DEP） domain-containing mammalian target of rapamycin （mTOR）-interacting protein （DEPTOR）， mTOR， and ribosomal protein S6 kinase 1 （S6K1） were detected using real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultsCompared to the normal group， the model group exhibited significantly decreased glucose consumption and glycogen content， increased levels of BCAAs and BCKAs， downregulated expression of BCKDH and DEPTOR， and upregulated mTOR and S6K1 expression （P<0.01）. In comparison to the model group， QRP treatment at all doses significantly enhanced glucose consumption and glycogen content while reducing BCAAs and BCKAs levels （P<0.01）. The high- and medium-dose QRP groups demonstrated significant upregulation of BCKDH mRNA transcription and protein expression， as well as DEPTOR mRNA transcription. Moreover， the DEPTOR protein expression level was significantly increased in high-， medium-， and low-dose QRP groups， while mTOR and S6K1 mRNA and protein expression levels were markedly downregulated （P<0.05， P<0.01）. Compared to the QRP + si-NC group， the QRP + si-BCKDH group exhibited increased BCAAs and BCKAs levels， significantly decreased BCKDH mRNA transcription and protein expression， downregulated DEPTOR mRNA and protein expression， and upregulated mTOR and S6K1 mRNA and protein expression （P<0.05， P<0.01）. ConclusionQRP may improve insulin resistance by reprogramming BCAAs metabolism. This effect involves upregulating BCKDH， reducing BCAAs and BCKAs levels， and suppressing the mTOR pathway activation.
