[Protective effect of bloodletting at twelve Well-points of hand on neurological function by regulating cortical microglia/macrophage polarization through the IL-10/STAT1 pathway in mice with traumatic brain injury].

OBJECTIVES: To observe whether pricking blood therapy at the twelve Well points of the hand (Well-point bloodletting) can regulate the cortical phenotypic polarization of microglia and macrophages via the interleukin-10 (IL-10)/signal transducer and activator of transcription 1(STAT1) pathway, suppress excessive neuroinflammation and neuronal apoptosis, and thereby alleviate neurological deficits induced by traumatic brain injury (TBI) in mice.

METHODS: Male C57BL/6 mice were used in the present study which is composed of 3 experiments. The TBI model was established by using an electronically controlled cortical impact device to perform cortical impact. Experiment 1:Twelve mice randomly selected were used as the sham-operation group. Twenty-four TBI mice were randomly and equally divided into a model and a Well-point bloodletting group. Mice in the Well-point bloodletting group received interventions at 6 h, 24 h and 48 h after TBI respectively. The modified neurological severity score (mNSS) and open field test were used to assess the mouse's neurological deficits and locomotor function. Immunofluorescence staining was used to detect the proportions of apoptotic neurons (NeuN+TUNEL+) and microglia (IBA1+) in the injured cerebral cortex tissues. Liquid chip assay was performed to measure the levels of inflammatory factors in the injured cortical tissues, and Western blot was used to detect the expression of cortical STAT1 protein. Experiment 2:The STAT1 agonist 2-NP was applied to activate STAT1 to verify whether STAT1 mediates the anti-inflammatory effect of Well-point bloodletting. Thirty-six TBI mice were randomly and equally divided into model, Well-point bloodletting and Well-point bloodletting+2-NP groups. Mice in the Well-point bloodletting+2-NP group received intraperitoneal injection of 2-NP 1 h before modeling and each bloodletting intervention. The mNSS was used to assess the neurological function, and flow cytometry was utilized to quantify the proportions of M1 microglia and macrophages in the injured cerebral cortex. Experiment 3:this part of the study is conducted to clarify the pivotal role of IL-10 in the anti-inflammatory effect of Well-point bloodletting. The successfully modeled TBI mice were randomly allocated to the model, Well-point bloodletting and Well-point bloodletting+IL-10 antagonist groups, with 10 mice in each group. Mice in the IL-10 antagonist group received injection of IL-10 neutralizing antibody into the lateral ventricle on the contralateral side of the injured cortex 1 h after each bloodletting intervention to block the endogenous IL-10. The neurological deficits were evaluated using mNSS. Western blot was used to detect STAT1 phosphorylation in the brain tissues, and the flow cytometry was applied to measure the proportions of M1 microglia and macrophages in the injured cerebral cortex tissues.

RESULTS: Well-point bloodletting significantly reduced the mNSS scores at 6 h and 48 h (P<0.01, P<0.05) after TBI, down-regulated the proportions of apoptotic neurons and microglia (P<0.05, P<0.01), STAT1 phosphorylation (P<0.05) and IL-2 content (P<0.05), and increased the total travel distance 48 h after TBI (P<0.01) and IL-10 content (P<0.01). Well-point bloodletting+2-NP administration eliminated the effects of bloodletting in down-regulating the mNSS score after TBI (P<0.01) and the proportion of M1 microglia (P<0.01). Well-point bloodletting+IL-10 antagonist administration abolished the effects of Well-point bloodletting in lowering the mNSS score at 6, 24 and 48 h after TBI (P<0.05, P<0.01), suppressing STAT1 phosphorylation (P<0.05) and in reducing the proportions of M1 microglia and M1 macrophages in the injured cerebral cortex (P<0.05).

CONCLUSIONS: Well-point bloodletting can improve the neurological deficits and motor function in TBI mice, which may be related to its functions in inhibiting M1 polarization of microglia and macrophages through the IL-10/STAT1 pathway, and mitigating cortical neuroinflammation and neuronal apoptosis.