["Shugan Tiaoshen" needling improves depression after ischemic stroke by regulating AMPK-dependent autophagy]

To observe the effect of "Shugan Tiaoshen"(soothing the liver and regulating the mental activities) needling on the expressions of AMP-activated protein kinase (AMPK) and autophagy-related proteins in the prefrontal cortex of rats with depression after ischemic stroke (post-stroke depression), in order to explore its mechanisms underlying improvement of post-stroke depression behaviors.

A total of 60 male SD rats were randomly divided into sham operation (control), stroke, post-stroke depression, inhibitor, acupuncture, and acupuncture + inhibitor groups. The post-stroke depression model was established by occlusion of the middle cerebral artery and reperfusion (MCAO/R), combined with chronic unpredictable mild stimulation (CUMS). Rats in the inhibitor group received bilateral lateral ventricular injection of AMPK inhibitor (0.1 μmol, 10 μL). Manual acupuncture stimulation was applied to "Baihui" (GV20), "Yintang" (GV24⁺), "Hegu" (LI4), and "Taichong" (LR3) for the acupuncture group, once daily for 14 d. Rats in the acupuncture + inhibitor group received acupuncture treatment after 3 d of AMPK inhibitor injection. Neurologic impairment was assessed by Longa score. Behavioral assessment of depression was conducted by sucrose preference test and open field test. H.E. staining was used to observe the morphological changes of prefrontal cortex. Western blot was used to detect the protein expression levels of AMPK, p-AMPK, Beclin1, P62 and LC3 in the prefrontal cortex tissue. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of prefrontal neurons.

Compared with the control group, only the total locomotor distance, the percentage of central area dwell time within 5 min and the p-AMPK/AMPK were significantly decreased (P<0.01) in the stroke group while Longa score was increased (P<0.01). Compared with the control group, the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, levels of p-AMPK/AMPK, Beclin1 and LC3Ⅱ/Ⅰ were significantly decreased (P<0.01, P<0.05), while Longa score and P62 expression was significantly up-regulated (P<0.01) in the post-stroke depression group. Compared with the post-stroke depression group, the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, and the levels of p-AMPK/AMPK, Beclin1, and LC3Ⅱ/Ⅰ were considerably increased (P<0.01), while P62 expression was notably decreased (P<0.05) in the acupuncture group. Comparison between the inhibitor group and the acupuncture+inhibitor group showed that the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, and the expression level of p-AMPK/AMPK were even lower in the former group than those in the latter group (P<0.05, P<0.01), and the expression level of P62 was higher in the former group than in the latter group (P<0.01). H.E. staining showed loose arrangement of prefrontal cortex neurons, reduced number, pyknotic and hyperchromatic nuclei, even with degeneration and necrosis of some neurons in the stroke, post-stroke depression and the inhibitor groups, while increase in neuronal number, with relatively complete structure and uniform stain observed in the acupuncture group. Results of TEM showed disrupted neuronal nuclear membrane, reduction in the number of mitochondria with moderate to severe swelling, and decrease or disappearance of the cristae, and transparent matrix with vacuolation in the post-stroke depression group, which was relatively milder (mild swelling, nearly normal structure and more autophagosome forming) in the acupuncture group and acupuncture+inhibitor group.

"Shugan Tiaoshen" needling can improve the depressive symptoms in post-stroke depression rats, which may be related to its function in activating AMPK-dependent autophagy process.