The Mechanisms Of Pseudoginsenoside GQ Improving Cognitive Impairment Induced By Chronic Intermittent Hypoxia In Mice By Regulating Microglia Polarization

Obstructive sleep apnea(OSA)is a sleep-related breathing disorder characterized by recurrent partial or complete upper airway obstruction,chronic intermittent hypoxia(CIH)is its important pathophysiological feature.Globally,it is estimated that 936 million adults(aged 30-69 years)have mild to severe OSA in the world.Cognitive dysfunction is one of the common complications of OSA,which is mainly manifested as the decline of attention,memory,alertness,construction ability and executive function.continuous positive airway pressure(CPAP)is currently one of the most effective therapies for OSA and has been shown to correct breathing disorders and the resulting transient oxygen desaturation,thereby reducing sleep fragmentation during sleep.However,the improvement of cognitive function in OSA patients treated with CPAP is still unclear,and the low compliance of CPAP also limits its popularization and application.Therefore,it is of great clinical significance to find effective therapeutic drugs for OSA-related cognitive impairment.Ginsenosides,the main active components of ginseng,have been widely used to treat a variety of diseases due to their anti-tumor,anti-inflammatory and antioxidation effects.Secondary ginsenosides produced after ginsenoside degradation have been found to exert more potent pharmacological effects in vivo.Pseudoginsenoside GQ(PGQ)is an octilon-type secondary ginsenoside synthesized from Rg3.Some studies have found that PGQ can improve acute myocardial infarction and adriamycin-induced myocardial injury,and is a therapeutic drug for myocardial ischemia and angina.However,whether PGQ can improve neuroinflammation and protect cognitive function and the underlying mechanisms remain unclear.The aim of this study is to investigate the effects and underlying molecular mechanisms of PGQ on neuroinflammation and cognitive impairment induced by CIH at human,animal and cellular levels.It is divided into the following four parts:1.The relationship between serum inflammatory factor levels and cognitive function in OSA patientsThe general data of suspected OSA patients who visited the Second Hospital of Jilin University from July 2022 to June 2023 were collected,including demographic data,montreal cognitive assessment(MOCA)data,polysomnogram(PSG)data and Epworth sleepiness score(ESS).The serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and HMGB1 were detected by ELISA.Spearman’s method was used to test the correlation between serum inflammatory factors and sleep parameters and Mo CA scores in OSA patients.Binary logistic regression model was established to analyze the independent risk factors affecting cognitive dysfunction in OSA patients.The results showed that the serum levels of TNF-α,IL-1β and HMGB1 in OSA patients increased gradually with the severity of OSA,and the Mo CA score of the moderate to severe OSA group was significantly lower than that of the normal control group,indicating that moderate to severe OSA can lead to cognitive dysfunction.In addition,The serum levels of TNF-α,IL-1β and HMGB1 in OSA patients were positively correlated with apnea-hypopnea index(AHI)and oxygen desaturation index(ODI)and the percentage of time spent with an Sp O2 below 90%(TS90%).It was negatively correlated with minimum oxygen saturation(Min Sp O2)and Mo CA score,but not correlated with mean oxygen saturation(Mean Sp O2)and ESS.Binary logistic regression analysis showed that ODI,TNF-α and HMGB1 were independent risk factors for cognitive dysfunction in OSA patients.2.Establishment of CIH-induced cognitive impairment model in miceAccording to the CIH cycle(2 weeks,4 weeks,6 weeks),C57BL/6 mice were randomly divided into four groups(n = 8 in each group): NC group,CIH2 W group,CIH4 W group and CIH6 W group.The CIH animal model was established by intermittent hypoxia chamber.Y maze and Morris water maze were used to evaluate the spatial learning and memory of mice.western blot and ELISA were used to detect the expression levels of TNF-α,IL-1β and HMGB1 in the hippocampus and serum,respectively.The results showed that the spatial learning and memory abilities of CIH4 W and CIH6 W mice were significantly reduced,while CIH2 W mice showed no significant difference.Compared with the control group,the expression levels of inflammatory factors in hippocampus and serum of CIH2 W,CIH4W and CIH6 W groups were significantly increased,and gradually increased with the prolongation of CIH cycle.These results suggest that CIH can cause neuroinflammation and peripheral inflammatory responses,and that CIH exposure for more than 4 weeks induces cognitive dysfunction in mice.3.Protective effects and mechanisms of PGQ on CIH-induced cognitive impairment in miceC57BL/6 mice were randomly divided into five groups: NC group,CIH group,CIH+PGQ5 mg/kg group,CIH+PGQ10 mg/kg group,and CIH+PGQ20 mg/kg group.PGQ or equal volume of solvent was injected intraperitoneally daily during the first three days of CIH exposure(4 weeks)and throughout the experimental period.Y maze and Morris water maze were used to detect the cognitive function of mice in each group.The apoptosis of hippocampal neurons was detected by HE staining,Nissl staining,TUNEL staining and western blot.Immunohistochemistry(IHC),western blot and ELISA were used to detect the activation of microglia in hippocampus and the expression levels of inflammatory factors in hippocampus and serum,respectively.Immunofluorescence staining and western blot were used to detect the expression of HMGB1,TLR4,p-NF-κB,NF-κB,p-IκB and IκB in hippocampus.To investigate whether PGQ improved CIH-induced cognitive dysfunction by regulating HMGB1/TLR4/NF-κB signaling pathway,CIH mice were treated with glycyrrhetinic acid(GLY),an inhibitor of HMGB1.The experiments were divided into three groups:CIH group,CIH+GLY group,and CIH+PGQ group.The activation of microglia in hippocampus,the expression of inflammatory factors in hippocampus and serum,neuronal apoptosis,and cognitive function were detected,respectively.The results showed that PGQ could significantly improve CIH-induced cognitive dysfunction,reduce hippocampal neuronal apoptosis,inhibit microglia M1 polarization and neuroinflammation,and regulate HMGB1/TLR4/NF-κB signaling pathway.The effects of GLY and PGQ are consistent,suggesting that HMGB1 plays an important role in CIH-induced cognitive dysfunction in mice.PGQ inhibits CIH-induced hippocampal microglia activation and subsequent neuorinflammation response and attenuates neuroinflammation-induced hippocampal neuronal apoptosis by downregulating the HMGB1/TLR4/NF-κB signaling pathway,thereby improving cognitive dysfunction in CIH mice.4.Protective effects and mechanisms of PGQ on IH-induced BV-2 microglial activation and neurotoxicityThe IH model of BV-2 microglia was constructed,and the anti-inflammatory and neuroprotective effects of PGQ in vitro were evaluated from the aspects of cell proliferation,inflammatory factors,microglia activation,intracellular reactive oxygen species(ROS)level,and apoptosis of HT22 neurons.The binding ability of PGQ to HMGB1 was predicted by molecular docking technology.Western blot,q RT-PCR,ELISA and immunofluorescence were used to detect the regulatory effect of PGQ on HMGB1/TLR4/NF-κB signaling pathway.GLY,an inhibitor of HMGB1,was used in BV-2 microglia to further verify whether PGQ inhibited IH-induced microglia M1 polarization and subsequent inflammatory mediator expression by regulating HMGB1/TLR4/NF-κB signaling pathway.The results showed that PGQ at the concentration of 20 μM had the most significant anti-inflammatory effect.PGQ could significantly inhibit IH-induced BV-2 cells M1 polarization and ROS production,and promote M2 polarization,thereby improving neuronal apoptosis induced by inflammatory mediators.Molecular docking technology showed a strong interaction between PGQ and HMGB1.PGQ inhibited IH-induced nuclear and cytoplasmic translocation and release of HMGB1,and decreased the expression of HMGB1,TLR4,p-NF-κB and p-IκB.The similar effects of GLY and PGQ suggest that HMGB1 plays an important role in IH-induced microglia M1 polarization and neuroinflammation.These results suggest that PGQ inhibits IH-induced BV-2 microglia M1 polarization by down-regulating HMGB1/TLR4/NF-κB signaling pathway,thereby reducing the toxic effect of inflammatory mediators on HT22 neurons,thus playing an indirect neuroprotective effect.In conclusion,this study first found the correlation between inflammatory factors and cognitive dysfunction in OSA patients from human clinical experiments.It was then confirmed PGQ ameliorated CIH-induced cognitive dysfunction in mice by inhibiting microglia M1 polarization and reducing neuroinflammation-induced neuronal apoptosis at the animal and cellular levels.Molecular docking technology showed a strong interaction between PGQ and HMGB1,and PGQ played a neuroprotective role by inhibiting HMGB1/TLR4/NF-κB signaling pathway to regulate microglial polarization.This may provide a natural and effective new drug candidate for the treatment of OSA-related cognitive impairment.