ObjectiveTo investigate the mechanism by which Guipitang regulates mitochondrial biogenesis of hippocampal neurons in methylmalonic aciduria （MMA） rats through the adenosine monophosphate-activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1α （AMPK/PGC-1α） signaling pathway.MethodsFifty 5-day-old Wistar rats were randomly divided into a blank group， a model group， a Guipitang group （9.3 g·kg-1）， an AMPK inhibitor group （2.5 mg·kg-1 Compound C）， and a Guipitang + AMPK inhibitor group （9.3 g·kg-1 + 2.5 mg·kg-1 Compound C） using a random number table method. After 3 weeks of administration， the Morris water maze test was used to assess the rats' learning and memory abilities. Hematoxylin-eosin （HE）， Nissl， and terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining was performed to detect histopathological changes and apoptosis in rat hippocampal tissue. Colorimetric assays were employed to measure mitochondrial adenosine triphosphate （ATP） content in hippocampal tissue. The mitochondrial superoxide red fluorescent probe method （MitoSOX Red） was used to assess mitochondrial reactive oxygen species （ROS） levels. The JC-1 assay was carried out to detect mitochondrial membrane potential in hippocampal tissue. Transmission electron microscopy （TEM） was conducted to observe pathological changes in mitochondria. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect mitochondrial DNA copy numbers. Western blot was employed to detect AMPK， PGC-1α， phosphorylated AMPK （p-AMPK）， nuclear factor E2-related factor 1 （Nrf1）， nuclear factor E2-related factor 2 （Nrf2）， and mitochondrial transcription factor A （TFAM） in rat hippocampal tissue.ResultsCompared with those in the blank group， rats in the model group exhibited more dispersed movement paths， were unable to locate the platform independently， and had prolonged escape latency， reduced frequency of crossing the platform， significantly reduced time spent in the target quadrant， and significantly increased time spent in the opposite quadrant （P<0.01）. Compared with those in the model group， rats in the Guipitang group and the Guipitang + AMPK inhibitor group had significantly shortened escape latency， significantly increased frequency of crossing the platform， significantly increased time spent in the target quadrant， and significantly reduced time spent in the opposite quadrant （P<0.05， P<0.01）. Compared with the Guipitang group， the AMPK inhibitor group had a longer escape latency， significantly reduced frequency of crossing the platform， significantly reduced time spent in the target quadrant， and significantly increased time spent in the opposite quadrant （P<0.01）. The model group showed disorganized arrangement of hippocampal neurons and nuclear pyknosis. After treatment with Guipitang， the number of regular neurons increased， while the improvement in the Guipitang + AMPK inhibitor group was less than that in the Guipitang group （P<0.01）. Electron microscopy showed that the cristae structure of mitochondria was extensively lost in the model group， but significantly recovered in the Guipitang group （P<0.01）. The Guipitang + AMPK inhibitor group still exhibited mitochondrial membrane rupture and reduced cristae structure. The apoptosis rate was significantly increased in the model group. Compared with the blank group， the model group had significantly increased ROS levels， significantly reduced ATP content， and significantly decreased mitochondrial membrane potential （P<0.01）. Compared with the model group， the Guipitang group had significantly reduced ROS levels， significantly increased ATP content， and significantly increased mitochondrial membrane potential （P<0.01）. Compared with the model group， the Guipitang group showed a significant decrease in apoptosis rate （P<0.01）， and this effect was partially reversed by the AMPK inhibitor （P<0.01）. The model group showed significantly reduced TFAM， PGC-1α， Nrf1， and Nrf2 mRNA expression and protein expression levels （P<0.01）. Compared with the model group， the Guipitang group showed significantly increased TFAM， PGC-1α， Nrf1， and Nrf2 mRNA expression and protein expression levels （P<0.01）. Compared with the blank group， the model group showed significantly reduced p-AMPK/AMPK protein expression levels （P<0.01）. Compared with the model group， the Guipitang group showed significantly increased p-AMPK/AMPK protein expression levels （P<0.01）.ConclusionGuipitang can improve cognitive dysfunction in rats by activating the AMPK/PGC-1α signaling pathway， promoting mitochondrial biogenesis， and alleviating pathological damage to hippocampal neurons.
BAI Huanghuang, ZHENG Hong, LU Xiangpeng
