Huatuo Zaizao Pills Alleviate Neuroinflammation and Neuronal Apoptosis via Microglial M2 Polarization in a Permanent...

This study aimed to explore the potential neuroprotective effects of Huatuo Zaizao pills (HTZZ) in modulating microglial polarization-mediated neuroinflammation and apoptosis.

Twenty-four male mice were included in the study. Sixteen mice underwent permanent middle cerebral artery occlusion (pMCAO) and were randomly assigned to either the MCAO group (vehicle, n = 8) or the MCAO+HTZZ group (HTZZ, 5.80 g/kg, n = 8). Eight mice underwent sham surgery and received vehicle treatment (Sham group). HTZZ or vehicle was administered orally for seven days before surgery and continued for three days after surgery. Neurological functions were evaluated using the Longa score, adhesive removal test, grid-walking test, and forelimb grip strength test. Neuronal damage was assessed via Nissl staining and double immunofluorescence staining for postsynaptic density protein 95 (PSD95) and microtubule-associated protein 2 (MAP2). We also quantified pro-inflammatory cytokines as well as markers of microglial polarization. Apoptosis was assessed through both TUNEL assays and Western blot quantification of key apoptosis-associated proteins.

When comparing HTZZ-treated and untreated ischemic animals at 72 h after pMCAO, mice receiving HTZZ exhibited notable functional improvement. HTZZ treatment suppressed pro-inflammatory cytokines and decreased GFAP- and Iba-1-positive cells in the peri-infarct area. Analysis of microglial phenotypes revealed that HTZZ diminished the expression of iNOS/Iba-1+ cells while increasing Arg-1/Iba-1+ cells (both P<0.05). HTZZ markedly lowered the proportion of TUNEL-positive profiles and shifted the balance of apoptosis regulators toward higher Bcl-2/Bax ratios.

These findings suggest that HTZZ may exert neuroprotective effects through modulation of microglial polarization and attenuation of neuroinflammatory responses. By shifting microglia/macrophages away from a pro-inflammatory phenotype and suppressing cytokine overproduction, HTZZ appears to mitigate secondary neuronal injury in the peri-infarct region. The observed reduction in apoptotic markers and restoration of the Bcl-2/Bax balance indicate HTZZ's potential role in inhibiting neuronal apoptosis. Notably, increased Akt phosphorylation was observed, indicating the possible involvement of the PI3K/Akt pathway.

HTZZ demonstrates potential neuroprotective effects in permanent cerebral ischemia, possibly through modulation of microglial polarization and attenuation of neuronal apoptosis. These findings support further investigation into HTZZ as a multi-target therapeutic candidate for ischemic stroke.