Mechanisms of acupuncture in inhibition of ferroptosis in intracerebral hemorrhage mice based on RNA m6A methylation sequencing

To observe the effect of acupuncture on N6-methyladenosine (m6A) methylation andferroptosis in brain tissue of mice with intracerebral hemorrhage (ICH), so as to explore its mechanisms underlyingpromoting neural function recovery.

Fifty-four male C57BL/6J mice were randomly divided into shamoperation, model and acupuncture groups, with 18 mice in each group. The ICH model was established by injection ofthe mouse's auto-blood (10 μL) into the right caudate nucleus. Mice of the acupuncture group received acupuncture at "Baihui" (GV20) for 30 min, twice daily for 7 days. The neurological functions (locomotor and balance deficits) wereevaluated according to wire hanging test (0—5 points) and balance walking test (0—5 points) before modeling andafter treatment. Histopathological changes in the brain were observed by HE staining. RNA-sequencing and m6A-sequencing were used to analyze the differential m6A modification genes. Immunofluorescence and Western blot wereused to detect the expression of glutathione peroxidase 4 (Gpx4) and sirtuin 3 (Sirt3) proteins in brain tissue, respectively.

HE staining showed that in the brain tissue nucleus was pyknotic, inflammatory cells wereinfiltrated, neurons were edematous and necrotic in the model group, which were relatively milder in the acupuncturegroup. The sequencing results showed that 592 differentially expressed genes were screened in the model group vs sham operation group, and 849 differentially expressed genes were screened in the acupuncture group vs model group. Five genes that inhibit ferroptosis were obtained by intersecting with genes that inhibit ferroptosis, namely charged multi vesicular body protein (Chmp) 1a, Chmp6, glutaredoxin (Glrx) 5, Gpx4, and Sirt3. Compared with the sham operation group, the wire hanging test and balance walking test scores, gene expression levels of Chmp1a, Chmp6, Glrx5, Gpx4, and Sirt3, Gpx4/NeuN and Sirt3/NeuN co-expressed fluorescence intensity, the protein expressions of Gpx4 and Sirt3 in brain tissue were all significantly decreased in the model group (P<0.01). Following the interventions, the wire hanging test and balance walking test scores, gene expression levels of Chmp1a, Chmp6, Glrx5, Gpx4, and Sirt3, Gpx4/NeuN and Sirt3/NeuN co-expressed fluorescence intensity, the protein expressions of Gpx4 and Sirt3 in brain tissue were all significantly increased in the acupuncture group compared with the model group (P<0.01, P<0.05).

Acupuncture can ameliorate neurological deficits, which may be associated with its function in improving m6A RNA modification and RNA expression in mice with ICH, and promoting the expression of ferroptosis antioxidation related proteins Gpx4 and Sirt3 in brain tissue.