P7C3-A20 Attenuates Microglial Inflammation And Brain Injury After ICH Through Activating The NAD~+/Sirt3 Pathway

Background Intracerebral hemorrhage(ICH)is an acute cerebrovascular disease with severe neurological impairment and high mortality.Pathologic processes following ICH include primary brain injury and secondary brain injury.Primary brain injury is the physical destruction of brain tissue caused by hematoma.It also triggers secondary brain damage,including inflammatory responses,oxidative stress,glutamate toxicity,calcium overload and blood-brain barrier damage.Therefore,it is imperative to research drugs that can alleviate secondary injury to improve the quality of life after ICH.Niacinamide adenine dinucleotide(NAD~+)is widely involved in many physiological activities,including cell energy synthesis,substance metabolism,DNA repair,gene expression,and aging.NAD~+ reduction was found to be associated with many neurological diseases,and NAD~+ supplementation may be a treatment strategy.P7C3-A20 is a novel chloropropyl carbazole compound that promotes NAD~+ endogenous synthesis by activating niacinamide phosphate ribosome transferase(NAD~+ synthetase).Previous studies have shown that P7C3-A20 significantly enhances NAD~+ synthesis.In a sense,the treatment of P7C3-A20,a drug that promotes endogenous NAD~+ synthesis,may be more effective and long-lasting than the direct administration of NAD~+ precursors.This study explored the NAD~+ changes and therapeutic effects of P7C3-A20 on ICH.In the pre-experiment,we found that P7C3-A20 strongly inhibited neuroinflammatory response after ICH by RNA transcriptome sequencing.Neuroinflammation plays a vital role in secondary brain injury following ICH.Currently,microglia are the primary cells mediating inflammation after ICH.Activation of microglia releases a wide range of inflammatory factors,triggers inflammatory responses,and induces infiltration of circulating immune cells,including macrophages and T cells,to accelerate secondary brain damage.Therefore,we further investigated the role of P7C3-A20 and its underlying mechanisms in microglia following ICH.Sirt3,an NAD~+-dependent protein deacetylase,plays an important role in regulating mitochondrial function.NAD~+ level is critical for Sirt3 protein activity and deacetylation.Mitochondrial damage has been shown in many studies to be a vital factor in the secondary neuroinflammatory response following ICH.It has also been found that activation of Sirt3 protects mitochondrial function and thus inhibits microglia-mediated inflammatory responses.Accordingly,we hypothesized whether NAD~+/Sirt3-mediated mitochondrial quality control is involved in P7C3-A20’s suppression of inflammatory responses after ICH.To answer these questions,the study was designed into five parts:Part Ⅰ Correlation between plasma NAD~+ level and the severity as well as the prognosis of ICHMethods Included were 76 acute ICH patients admitted to Air Force Medical University Hospital No.2 between June 2021 and October 2022.NIHSS scores were assessed upon admission,and disease severity was classified into mild to moderate(NIHSS score ≤ 15)and moderate to severe(NIHSS > 15).The mRS score was also assessed,and prognostic function was classified into favorable prognosis(mRS 0-2 score)and poor prognosis(mRS 3-6 score).Plasma NAD~+ levels were detected in each group,and the correlation of NAD~+ with NIHSS and mRS scores was analyzed.We also evaluated the prognostic value of NAD~+ for ICH severity and prognosis.Logistic regression was used to analyze the relevant factors affecting ICH outcomes.Results 1.A total of 76 ICH patients were included with a mean plasma NAD~+ of 24.67 ± 10.80 μg / mL.Plasma NAD~+ levels were significantly higher in patients with mild to moderate severity(t = 8.26,P < 0.05);Plasma NAD~+ levels were significantly higher in patients with favorable outcomes than in patients with poor outcomes(t = 7.20,P < 0.0001).2.Spearman correlation analysis showed that plasma NAD~+ levels were negatively correlated with NIHSS scores(r =-0.59,P < 0.05)and mRS scores(r =-0.66,P < 0.01)in ICH patients.3.ROC analysis of plasma NAD~+ levels to diagnose ICH severity: AUC 0.91 [95% CI(0.828,0.967)],cut-off 19.5 μg/mL,sensitivity 90.48%,specificity 87.27%.ROC analysis of plasma NAD~+ levels to diagnose ICH patient outcome: AUC 0.88,[95% CI(0.789,0.944)],cut-off 20.3 μg/mL,sensitivity 89.4%,specificity 75.9%.4.The single-factor analysis found that NIHSS,ICH volume,blood sugar,APTT and NAD~+ levels all affected prognosis.Taking into account these confounding variables,the multifactorial logistic analysis revealed an OR of 0.33(95% CI 0.73-0.99)for plasma NAD~+ levels,suggesting that reduced NAD + levels are independent risk factors for prognosis.Conclusion Plasma NAD~+ levels are associated with the severity and prognosis of ICH,which is valuable in diagnosing severity and outcome in ICH patients.Besides,NAD~+ deficiency is an independent risk factor for prognosis.Part Ⅱ P7C3-A20 relieves secondary brain injury after ICHMethod Healthy adult male C57BL/6 mice were randomly divided into sham group,ICH group and three subgroups(ICH+P7C3-A20)treated with different drug doses(5 mg/kg,10 mg/kg, 20 mg/kg P7C3-A20).In the ICH group,0.075 u collagen Ⅶ was injected into the striatum of mice using a stereo-positioning needle after skull drilling,while in the sham group,the same amount of saline was injected,and the rest of the procedure was the same.The ICH+P7C3-A20 group was injected intraperitoneally with different doses of P7C3-A20 immediately 30 min after ICH in mice.The optimal dose was determined by evaluating the neurobehavioral experimental results of each group.Brain edema,BBB and lesion volume were measured by brain water content,evens blue and MR.Western blot and immunofluorescence were used to detect apoptosis of nerve cells in each group.Result 1.In contrast to the sham group,the ICH group significantly increased the rate of footfault(P < 0.05),reduced the times of the affected forepaw use(P < 0.05),reduced the stay on the accelerated rotating rod(P < 0.05),and extended time needed in adhesive paper removal.10 mg/kg and 20 mg/kg P7C3-A20 exhibited more noticeable improvement in the sensorimotor ability of mice after ICH than 5 mg/kg P7C3-A20,with statistical significance.Notably,there was no significant difference between the effects of 10 and 20 mg/kg P7C3-A20 after ICH.So,according to above results,10 mg/kg P7C3-A20 was used as the lowest therapeutic dose for subsequent experiments.2.Compared to the ICH group,the ICH + P7C3-A20 group reduced lesions volume,brain water content and blood-brain barrier permeability,suggesting that P7C3-A20 treatment could reduce ICH volume and brain edema,and protect the integrity of the BBB after ICH.3.Western blotting displayed an evident rise of Bax and fall of Bcl2 in the ICH group compared to sham group,whereas both were significantly reversed by additional P7C3-A20 treatment.Meanwhile,TUNEL staining showed that compared to the sham,the ICH group significantly increased the levels of positive cells,while the P7C3-A20 treatment repressed cell apoptosis,suggesting that P7C3-A20 inhibited apoptosis of neurons after ICH.Conclusion P7C3-A20 significantly inhibited neuronal apoptosis,reduced the lesion volume,and decreased brain edema,thereby improving neurological function after ICH in mice.Part Ⅲ P7C3-A20 reduces microglial-mediated inflammatory damage after ICHMethod First,C57BL/6 mice were grouped and treated as described above.Differential gene expression was detected by RNA transcriptome sequencing and analyzed using GO and KEGG databases.Inflammatory cytokine m RNA levels were measured using qPCR.Microglia were observed by immunofluorescence staining.Result 1.The volcano plot showed that compared to the sham group,the ICH group contained 2303 upregulated and 309 downregulated genes,and that the ICH+P7C3-A20 group contained 222 upregulated and 1060 downregulated genes,compared to the ICH group.Venn diagram showed that the expression of 1047 overlapped genes was reversed by P7C3-A20 treatment.GO and KEGG analysis revealed critical biological processes enriched in the inflammatory response,suggesting that P7C3-A20 might regulate inflammatory response to exert its protective effect.qPCR results showed that P7C3-A20 treatment significantly reduced NFκB2,Nlrp3,IL-1b,and Ccl2 transcription levels.2.IBA1 and CD68 fluorescence staining showed that P7C3-A20 significantly reduced activated microglia’s number and fluorescence intensity,suggesting that PC3-A20 treatment significantly inhibited microglia activation after ICH.3.Using qPCR to detect microglia phenotype-specific markers,we found that,compared to the Sham group,ICH group displayed an evident rise of the M1 phenotype-specific markers(CD11b and i NOS)and fall of M2 phenotype-specific markers(CD206 and Arg-1)while ICH + P7C3-A20 group reversed this trend,suggesting that P7C3-A20 regulates microglia differentiation.Conclusion P7C3-A20 inhibits post-ICH microglia activation,thereby reducing post-ICH secondary inflammatory damage.Part Ⅳ P7C3-A20 attenuates mitochondrial damage in microglia by activating NAD~+ / Sirt3 pathwayMethod In cultured BV2 cell lines,we used OxyHb to stimulate BV2 cells 24 h to mimic the ICH group,and 60 μM P7C3-A20 incubated two hours prior to OxyHb stimulation to mimic ICH+P7C3-A20 group.Sirt3-knockout BV2 cell lines(Sirt3 KO)were constructed using CRISPR/Cas9 gene editing.Wild-type BV2 cell lines were divided into three groups: Vehicle,OxyHb,and OxyHb+P7C3-A20,and Sirt3 KO cell lines were also grouped as described above.Mitochondrial morphology,reactive oxygen species(ROS)levels and mitochondrial membrane potential(MMP)levels were detected using confocal microscopy;ATP levels in cells and tissue using the ATP assay kit;mitochondrial complex I activity in cells and tissues using the ATP assay kit.The conditioned gene knockout(CKO)mice were constructed using Cre/Lox P,and divided into Sham,ICH,and ICH+P7C3-A20,as wildtype mice were grouped.Microglia mitochondria were detected by electron microscopy.Result 1.In vitro of wild-type cell lines,we found that P7C3-A20 treatment significantly inhibited mitochondrial fragmentation induced by OxyHb,indicating that P7C3-A20 could maintain mitochondrial morphology integrity after ICH.Meanwhile,the indicators of mitochondrial function showed that ROS generation increased markedly,while MMP,ATP levels and mitochondrial complex I activity decreased significantly in the OxyHb group compared to that in vehicle group.Whereas these changes were revered by P7C3-A20 treatment,suggesting that P7C3-A20 could alleviate mitochondrial dysfunction after ICH.2.In wild-type BV2 cell lines,P7C3-A20 treatment significantly increased NAD~+ levels.In Sirt3 KO BV2,P7C3-A20 also reversed NAD~+ depletion after OxyHb treatment,indicating P7C3-A20 could also mediate the enhancement of NAD~+,which was not affected by Sirt3.3.We further found that the protective effect of P7C3-A20 on OxyHb-induced mitochondrial morphology damage and dysfunction was counteracted in knockout groups compared to non-knockout groups.These results indicated that P7C3-A20 alleviated mitochondrial morphological damage and protected mitochondrial function in a Sirt3-dependent manner.4.The results of electron microscopy showed that ICH group feature mitochondrial swelling,mitochondrial membrane disruption and loss of mitochondrial cristae,which are relieved by additional P7C3-A20 treatment.While compared with control littermates,the protective effect of P7C3-A20 on mitochondrial ultrastructure was partially suppressed in Sirt3 CKO mice.Meanwhile,the indicators of mitochondrial function showed that P7C3-A20 exerted protective effects on those indicators in Sirt3fl/fl mice after ICH,but not in Sirt3 CKO mice.These results suggested that Sirt3 mediates the role of P7C3-A20 in protecting mitochondrial morphology and function in post-ICH microglia in mice.Conclusion P7C3-A20 protected mitochondrial morphology and function in microglia by activating NAD~+/Sirt3 deacetylase activity after ICH.Part Ⅴ P7C3-A20 decreases microglial inflammation and secondary damage after ICH through the NAD~+/Sirt3 pathwayMethod The mice were grouped and treated as described in part IV.Microglia activation and inflammatory factors were detected by immunofluorescence and qPCR.Finally,brain edema,BBB permeability and neurobehavioral function were measured.Result 1.IBA1/CD68 staining showed that P7C3-A20 significantly reduced the level of activated microglia compared with the ICH group in Sirt3fl/fl mice,while we did not observe the same protective effect of P7C3-A20 in Sirt3 CKO mice.Meanwhile,P7C3-A20 significantly suppressed the expression of NFκB2,Nlrp3,IL-1b,Ccl2 at the transcriptional level compared to ICH group in Sirt3fl/fl mice,but not in Sirt3 CKO mice.These results indicated that Sirt3 CKO in the microglia countered the inhibitory effect of P7C3-A20 on neuroinflammation in mice.2.The results of the water content and Evens blue content showed that the inhibitory effect of P7C3-A20 on BBB impairment measured was also partially suppressed in Sirt3 CKO mice compared with that in Sirt3fl/fl mice.We also found the same evidence in brain water content that P7C3-A20 failed to alleviate ICH-induced brain edema in Sirt3 CKO mice,but could alleviate the brain edema in Sirt3fl/fl mice.These results suggest that Sirt3 mediates the role of P7C3-A20 in reducing BBB damage and brain edema after ICH.In neurobehavioral experiments,we did not observe apparent inhibitory effects of P7C3-A20 on ICH-induced neurological deficits in Sirt3 CKO mice,compared to those protective effects in Sirt3fl/fl mice.These results indicated that microglial Sirt3 knockout countered the protective effects of P7C3-A20 for neurological recovery after ICH.Conclusion P7C3-A20 suppressed microglial-mediated inflammatory damage,protected the integrity of the BBB,relieved cerebral edema,and improved neurological function after ICH through activating the Sirt3 pathway.Our study highlights the important role of Sirt3-mediated mitochondrial quality control in P7C3-A20’s neuroprotection and provides important targets and ideas for drug development.