Effects of Zhu Lian's type Ⅱ inhibition needling on cerebral angiogenesis in rats with ischemic stroke based on the Ang-1/Tie-2 pathway

To observe the effects of Zhu Lian's type Ⅱ inhibition needling on neurological function and angiogenesis in rats with ischemic stroke (IS), so as to explore its potential mechanisms.

Wistar rats were divided into sham operation group, model group, acupuncture group, and acupuncture+angiogenin (Ang)-2 group, with 12 rats in each group. A suture method was used to establish the IS model. The acupuncture group received Zhu Lian's type Ⅱ inhibition needling at the "Guanyuan" (CV4), "Guilai" (ST29), "Zusanli" (ST36), "Sanyinjiao" (SP6), and "Fenglong" (ST40) acupoints for 30 min per session. Rats in the acupuncture+Ang-2 group were injected via the tail vein with Ang-2 recombinant protein (10 mg/kg) before Zhu Lian's type Ⅱ inhibition needling intervention. All treatments were administered once daily for 7 consecutive days. Neurological deficit scores were assessed in rats of each group. TTC staining was employed to determine the percentage of cerebral infarction volume. HE staining was used to observe the pathological changes in the ischemic penumbra cortex. Immunohistochemistry was used to evaluate microvascular density in the ischemic penumbra cortex. Western blot was conducted to detect the expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), Ang-1, Ang-2, tyrosine kinase 2 (Tie-2), and phosphorylated Tie-2 (p-Tie-2) proteins in the ischemic penumbra cortex.

Compared with the sham operation group, the model group showed elevated neurological deficit scores, cerebral infarction volume percentage, microvascular density in the ischemic penumbra cortex, and protein levels of VEGF, VEGFR-2, Ang-1, and p-Tie-2/Tie-2 ratio. Conversely, Ang-2 protein level was decreased (P < 0.05). Compared with the model group and the acupuncture+Ang-2 group, rats in the acupuncture group had lower neurological deficit scores, cerebral infarction volume percentage, and Ang-2 protein level. While microvascular density in the ischemic penumbra cortex and the protein levels of VEGF, VEGFR-2, Ang-1, and the p-Tie-2/Tie-2 ratio were further increased (P < 0.05). In the model group, the number of neurons in the ischemic penumbra cortex of rats was decreased, the nuclei were shrank or disappeared, the cell bodies were vacuolated and degenerated, accompanied by a large number of inflammatory cell infiltrations. Compared with the model group, the injury of cortical neurons in the acupuncture group was alleviated. Compared with the acupuncture group, the injury of cortical neurons in the acupuncture+Ang-2 group was aggravated.

Zhu Lian's type Ⅱ inhibition needling can improve neurological function in IS rats, possibly by activating the Ang-1/Tie-2 pathway to promote angiogenesis.