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Effect of Zuogui Jiangtang Tongmai Prescription on Astrocyte Injury by AGEs Combined with Oxygen-glucose Deprivation Based on AMPK/mTOR/ULK1 Pathway Related to Autophagy / 中国实验方剂学杂志
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 90-99, 2022.
Article in Chinese | WPRIM | ID: wpr-940765
ABSTRACT
ObjectiveTo explore the effect and mechanism of Zuogui Jiangtang Tongmai prescription (ZGJTTMP) on astrocytesASs) injured by advanced glycation end products(AGEs) combined with oxygen-glucose deprivation (OGD). MethodCell counting kit-8 (CCK-8) was used to determine the optimal concentration of AGEs and the action time of OGD, and the optimal blood concentration of ZGJTTMP was selected for follow-up experiments. ASs were divided into normal group, model group (AGEs + OGD), ZGJTTMP group, an adenosine 5'-monophosphate-activated protein kinase (AMPK) inhibitor (Compound C) group, AMPK activator (AICAR) group, and combination group (ZGJTTMP + AICAR). The morphological changes in ASs in each group were observed under an inverted microscope. The cell survival rate in each group was detected by CCK-8. The content of interleukin-1β(IL-1β), interleukin-6IL-6), and tumor necrosis factor-α(TNF-α) was detected by enzyme-linked immunosorbent assayELISA). The number of autophagosomes in each group was counted under an electron microscope. The expression of microtubule-associated protein light chain 3 (LC3) was observed by immunofluorescence. The protein expression of LC3, p62, p-AMPK, AMPK, p-mammalian target of rapamycin (mTOR), mTOR, p-UNC-51 like kinase 1 (ULK1), and ULK1 was detected by Western blot. ResultAccording to the results of cell survival rate, 200 mg·L-1 AGEs and OGD for 6 h were selected as the optimal modeling conditions for the model group, and 5% was selected as the optimal blood concentration of ZGJTTMP. Under the inverted microscope, the cells were severely damaged after modeling, but the cell injury in the ZGJTTMP group and the Compound C group was significantly improved. As revealed by ELISA results, the content of IL-1β, IL-6, and TNF-α in the model group increased (P<0.01), and the content of inflammatory factors in the ZGJTTMP group and the Compound C group decreased (P<0.01). Under the electron microscope, the number of autophagosomes in the model group increased significantly. The immunofluorescence results showed that the expression area of LC3 increased in the model group (P<0.01), and the ZGJTTMP group and the Compound C group showed decreased number of autophagosomes and reduced expression area of LC3 (P<0.01). As demonstrated by the results of Western blot, compared with the normal group, the model group showed increased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK (P<0.01) and decreased p62, p-mTOR/mTOR, and p-ULK1/ULK1 (P<0.01). Compared with the model group, the ZGJTTMP group and the Compound C group showed decreased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK (P<0.01) and increased p62, p-mTOR/mTOR, and p-ULK1/ULK1 (P<0.01). ConclusionZGJTTMP possesses a protective effect on ASs with inflammatory injury by AGEs combined with OGD, which may be achieved by inhibiting the activation of the AMPK/mTOR/ULK1 pathway related to autophagy, thus inhibiting the overexpression of autophagy.

Full text: Available Index: WPRIM (Western Pacific) Type of study: Prognostic study Language: Chinese Journal: Chinese Journal of Experimental Traditional Medical Formulae Year: 2022 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Type of study: Prognostic study Language: Chinese Journal: Chinese Journal of Experimental Traditional Medical Formulae Year: 2022 Type: Article