| Objective: Obstructive sleep apnea hypopnea syndrome(OSAHS)is characterized by repeated collapse of the upper airway during sleep,and could eventually lead to chronic intermittent hypoxia(CIH)and sleep disorders.CIH is the most important pathological and physiological process of OSAHS.As a systemic disease,it can cause damage to multiple organ functions,among which the brain damage has recently been receiving heated attentions.OSAHS is associated with many neurological diseases,such as stroke,spinal cord injury and neurodegenerative changes.The pathological process of CIH might play an important role in the pathological mechanism of neurological injury.However,these mechanisms are still yet to clear.It has been reported that CIH could inevitably lead to the abnormal neurological functions by inducing neuronal apoptosis,astrocyte reactive hyperplasia and brain edema.Astrocytes are the main supporting trophic cells in the brain,supporting the survival of nerve cells,therefore targed inhibiting the transformation of astrocytes into pro-inflammatory phenotypes may provide neuroprotective effects for brain injury.Aquaporin-4(AQP4)was reported as an important water transport protein in the brain cells,which could not only regulate the cerebral water balance under pathological and physiological conditions,but also has a close relationship with neuroinflammation.Considering that CIH can cause cerebral edema and reactive astrocyte proliferation,and AQP4 is mainly expressed on the foot processes of astrocytes,it can be speculated that the changes in AQP4 expression might be related to CIH-induced brain injury,However,there have been no reports on brain injury induced by CIH and AQP4.p38 MAPK signaling pathway plays an important role in the regulation of neuroinflammation in the nervous system,and may be involved in the regulation of the biological function of astrocytes to pro-inflammatory type.Moreover,the activation of p38 MAPK signaling pathway was observed closely related to the expression of AQP4 in astrocytes.Previous studies have shown that the p38 MAPK pathway is involved in the expression of IL1-?and TNF-?-induced AQP4,thereby affecting cerebral edema after traumatic brain injury.MAPK family was proved selectively activated in a variety of hypoxic environments,and OSAHS-induced intermittent hypoxia could activate the p38 MAPK signaling pathway,which can cause damage to neurons in the hippocampus.We speculate that brain edema and brain injury induced by CIH may be related to the activation of p38 MAPK and AQP4,which has not been reported yet and need further study.Curcumin is a natural polyphenol compound found in plant turmeric,which plays anti-oxidative,anti-apoptotic and anti-inflammatory roles.In addition,curcumin can penetrate the blood-brain barrier and has a good protective effect on the nervous system.Recent studies have shown that curcumin can reduce cerebral edema by regulating the expression of AQP4 in various brain injury models such as cerebral trauma,cerebral ischemia-reperfusion,cerebral hemorrhage and chronic obstructive pulmonary disease(COPD).Evidences proved that curcumin could modulate the activation of p38 MAPK pathway in astrocytes.However,the mechanism of curcumin in CIH-related brain injury remain mysterious.We speculate that AQP4 and p38 MAPK signaling pathways may be involved in the protective effect of curcumin in CIH induced brain injury,which needs to be further confirmed In this work,CIH mice model was constructed to simulate the hypoxic characteristics of CIH pathology in vivo.The inflammatory response and pathophysiological changes of brain injury induced by CIH in mice were observed dynamically.The protective effect of curcumin on CIH-associated brain injury was evaluated to explore whether p38 MAPK signaling pathway and AQP4 are involved in the protective mechanism of curcumin against brain injury.Furthermore,in vitro experiments were conducted to further investigate the role of p38MAPK/AQP4 signaling pathway in hypoxic-induced astrocyte injury and the protective mechanism of curcumin,so as to explore whether the protective effect of curcumin on brain injury in CIH mice was partially achieved by regulating the activity of p38MAPK/AQP4 pathway.Methods:1.Dynamic observation of mice brain injury induced by CIH According to the random number table method,a total of 144 specific pathogen free(SPF)Balb/c mice were randomly divided into 2 main groups,including normoxia control group(n=72)and CIH group(n=72).CIH animals were placed in hyperbaric hyperxia chamber,in which oxygen concentration was set to 5%-21% alternately.Analternate cycle was 180 seconds,including a hyperxia treatment for 50 seconds and a normoxia treatment for 50 seconds.The cycle model guaranteed the minimum oxygen concentration(5%),the maximum oxygen concentration(21%)and 20 times of hypoxia-normoxia cycle per hour.The experiment was performed between 9:00 a.m.to5:00 p.m for eight consecutive hours a day.The observation was attained after 0 week,4weeks,8 weeks and 10 weeks.At the end of the observation point,the dynamic evolution of brain injury in CIH mice were explored by systematic inspection.The general condition of mice were observed by mobile activity,weight and the wet/dry(W/D)ratio of brain.Serum cortisol concentration was detected by corticosterone detection kit.Expression of TNF-? and IL1-? content in mice brain tissue were detected by enzyme linked immunosorbent assay(ELISA).Investigation of mouse cortex was processed by hematoxylin and eosin(H&E)staining method.Cell damage and neuron apoptosis of were evaluated by terminal dUTP-biotin nick end-labeling(TUNEL)staining.Glial fibrillary acidic protein(GFAP)immunohistochemical staining method was applied to investigate changes of morphology and GFAP expression in astrocytes.2.Effects and mechanisms of curcumin on chronic intermittent hypoxia induced brain injury in mice According to the random number table method,a total of 90 SPF Balb/c mice were randomly divided into 5 groups,including normoxic control group(n=18),CIH group(n=18),CIH + curcumin(50 mg/kg)group(n=18),CIH + curcumin(100 mg/kg)group(n=18)and CIH + curcumin(200 mg/kg)group(n=18).CIH mouse model was constructed according to the method mentioned in the first part.The mice were treated under CIH condition for 10 weeks.Each CIH group received different doses of curcumin(0,50,100,or 200mg/kg)by intragastric administration before daily intermittent hypoxia.At the end of the observation point,the proctective effect of curcumin to CIH-induced brain injury was evaluated.The general condition of mice were observed by mobile activity,weight and the W/D ratio of brain.Serum cortisol concentration was detected by corticosterone detection kit.Expression of TNF-? alpha and IL1-? content in mice brain tissue were detected by ELISA.Investigation of mouse cortex was processed by H&E staining method.Cell damage and neuron apoptosis of were evaluated by TUNEL staining.GFAP immunohistochemical staining method was applied to investigatechanges of morphology and GFAP expression in astrocytes.Moreover,the gene and protein expression level of AQP4 were detected by western blot and real-time fluorescence quantitative polymerase chain reaction(qPCR).Western blot experiments were performed to further elucidate the role of AQP4 and p38 MAPK pathways in the protective mechanism of curcumin against CIH-induced brain injury by detecting key proteins in p38 MAPK signaling pathways,including MAPK kinase 3(MKK3),phosphorylated MAPK kinase 3(p-MKK3),MAPK kinase 6(MKK6),phosphorylated MAPK kinase 6(p-MKK6),p38 MAPK,phosphorylated p38 MAPK(p-p38 MAPK)and C/EBP-homologous protein(CHOP).In addition,the correlation between AQP4 and p-p38 MAPK expression in astrocytes was observed by immunofluorescence double staining.3.The role of p38 MAPK/AQP4 signaling pathway in hypoxic-induced astrocyte injury and the protective mechanism of curcumin Primary astrocytes were used as the model cells to firstly isolate and purify the primary astrocytes.Fluorescence staining GFAP was used to detect the cell purity,and the purity of >90% could be used for subsequent experiments.The trial was divided into six groups,including three normoxic and three hypoxic groups: normoxic control group(Control),normoxia +curcumin group(Control+Cur)and normoxia + p38 inhibitor group(Control+p38inhibition);Hypoxia control group,Hypoxia+curcumin group(Hypoxia+Cur)and Hypoxia+p38 inhibitor group(Hypoxia+ p38inhibition).The cells were dosed and hypoxic(1% O2)according to the groups..Then cells were collected for subsequent detection after 3 hours culture.The lactic dehydrogenase(LDH)content in cell culture supernate was detected to evaluate the degree of cell damage by using LDH kit.Expression level of AQP4 in astrocytes was evaluated by immunofluorescence double staining,western blot and qPCR.Expression of p38 MAPK and p-p38 MAPK in protein level were detected by immunofluorescence double staining and western blot.The expression changes of AQP4,p38 MAPK and p-p38 MAPK were analyzed,and effect of curcumin on the expression of p38 MAPK and AQP4 in hypoxic-induced astrocyte injury were further investigated.Results:1.Dynamic evolution of brain injury induced by CIH in miceCIH-induced brain injury model in mice was successfully established by intermittent hypoxia in hypoxia chamber.At different time points of 4 weeks,8 weeks and 10 weeks after CIH exposure,CIH mice showed different characteristics: they were listless and showed obvious dullness in response to external stimuli.With the prolongation of CIH time,the CIH mice's mental listlessness,obvious dullness in response to external stimuli,reduced ability to resist external resistance and reduced ability to exercise was aggravated.Moreover,the average weight of CIH mice was significantly lower than that of mice of the same age(P<0.05).The concentration of serum cortisol and the levels of brain inflammatory factors TNF-? and IL1-? were gradually increased,reaching a peak at 8 weeks.However,no statistical difference between 10 weeks and 8 weeks was observed.the W/D ratio of brain increased gradually with the exposure time of CIH(P<0.05).Although there was no significant stastical difference between the two controlled groups at 4 weeks,the noted statistical difference was detected at 8 weeks and10 weeks,and severity of brain edema peaked at 10 weeks.The morphology of astrocytes was observed by H&E staining.The normoxic control group was observed with normal neuron structure,clear structure of cell nucleus and nucleoli.In the meantime,astrocytes in CIH group show gradually morphological changes with the exposure of CIH: concentrated and irregular nucleoli emerged in 4 weeks group,vacuoles that began to appear and increase gradually in the nucleus were observed in the8 week group,and typical vacuolar formation and surrounding inflammatory cell infiltration in nucleus were observed in the 10 weeks group.The results indicated that with the exposure of CIH,astrocytes showed different degrees of damage and cell edema,gradually aggravated pathological state and increased number of injured neuron cells.In the other hand,TUNEL assay showed that the number of apoptotic-positive cells in CIH group was significantly increased,compared with that in the normoxic control group at the same age(P<0.05).Also,the apoptosis degree increased with the increase of CIH time,significant statistical difference between the two groups were observed.Furthermore,with the prolongation of CIH time,astrocytes presented gradually morphological changes,showing typical characteristics of glial cell reactive hyperplasia such as cell body enlargement and thickening of foot processes.Further semi-quantitative analysis confirmed that the expression level of GFAP increased significantly with the extension ofCIH time,compared with the control group.Results in 4 weeks,8 weeks and 10 weeks were statistically significant in difference(P<0.05).2.p38 MAPK signaling pathway and AQP4 are involved in the protective effect ofcurcumin on CIH-induced brain injury Curcumin intervention group can significantly improve the general state of CIH mice,delay weight gain,and down-regulate the serum cortisol concentration,the expression of brain tissue inflammatory factors TNF-? and IL1-?,and the W/D ratio of brain.More significant dose dependence was observed in the medium and high curcumin concentration group.It was also observed that curcumin reduced the cell damage and prevented the neuron apoptosis in the cerebral cortex,brainstem and cerebellum by inhibiting the reactive proliferation of astrocytes and the upregulation of AQP4 expression.Moreover,the above changes have a dose-dependent relationship with the dose of curcumin.The results of western blot indicated that CIH group could significantly up-regulate the expression levels of phosphorylated protein(p-MKK3,p-MKK6,p-p38MAPK)of MKK3 and p38 MAPK in the brain tissues of mice,while the total protein levels of p38 MAPK,MKK3 and MKK6 were not significantly different between the groups.Curcumin intervention(100 and 200mg/kg)can inhibit its phosphorylation level,which is reflected in the down-regulation of p-MKK3,p-MKK6,p-p38 MAPK protein expression levels,and has a certain dose dependence on curcumin.In addition,it was also observed that the high-dosed curcumin treatment group could inhibit the CIH-induced overexpression of GFAP-AQP4 and GFAP-p-p38 MAPK double-labeled astrocytes in the cerebral cortex,cerebrum and brainstem by using immunofluorescence staining.3.The role of p38 MAPK/AQP4 signaling pathway in hypoxic-induced astrocyteinjury and the protective mechanism of curcumin Under hypoxic conditions,LDH concentration in cell culture medium was increased,while both P38 blocker group and curcumin group could inhibit the increase of LDH concentration.Hypoxia can induce the up-regulation of p-p38 mapk expression in astrocytes.P38 MAPK blockers successfully block the up-regulation of p-p38 mapk expression,while curcumin can inhibit the hypoxia-induced phosphorylation of p38 MAPK and down-regulate the expression of p-p38 mapk protein.In the meantime,increased expression of AQP4 at the protein and gene levels were also detected under hypoxia stimulation,and the expression were relatively decreased after the successful knockdown of the CIH-induced expression of p-p38 MAPK,indicating that hypoxia stimulation on astrocytes could activate the p38 MAPK/AQP4 signaling pathway.Interestingly,curcumin also inhibited p38 MAPK phosphorylation and AQP4 expression in hypoxia-treated astrocytes.However,no stastical significant changes were observed in controlled groups.Conclusion:1.CIH exposure could induce stress state of mice(slow weight gain and elevated serum cortisol concentration),chronic inflammatory lesions(upregulation of brain inflammation factor and emergence of cerebral edema)and structural change of brain tissue(neuronal injury and apoptosis of neurons and reactive proliferation of astrocytes).The phenomena were gradually aggravated with the extension of CIH time.2.Curcumin played a protective role in CIH-induced brain injury by alleviating the stress state of CIH-treated mice,reducing chronic inflammatory injury in brain tissue and attenuating structural changes in brain tissue.Moreover,this protective effect was reflected in multiple parts of cerebral cortex,cerebellum and brainstem,and there was a dose-effect relationship with curcumin concentration.3.The protective effect of curcumin on CIH-induced brain injury was proved to be related to the inhibition of p38 MAPK signaling pathway and downregulation of AQP4 expression.In vitro experiments have verified that curcumin can inhibit the p38MAPK/AQP4 pathway and reduce the hypoxic-induced primary astrocyte injury,so it is speculated that the protective effect of curcumin on brain injury in CIH mice may be partially achieved by inhibiting the activity of the p38MAPK/AQP4 pathway... |
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