| Vascular cognitive impairment(VCI)is a group of syndromes that ranges from mild cognitive impairment to dementia,caused by cerebrovascular diseases or risk factors.At present,clinicians judge the diagnosis and prognosis of VCI through imaging,cognitive function score,and underlying diseases.The incidence of VCI is increasing year by year.And VCI brings heavy family and social burdens.VCI presents a progressive course,there is no specific treatment at present,and early diagnosis and early intervention of related risk factors can effectively avoid or delay the occurrence of dementia.Most of the causes of vascular cognitive impairment are related to vascular factors,of which subcortical small vessel disease(SSVD)is a critical subtype of VCI.SSVD refers to a class of lesions involving subcortical blood vessels,the clinical manifestations are comprehensive cognitive decline,movement deficiency,mood disorders,and functional disabilities.It is characterized by white matter lesions(WMLs),lacunes,and cerebral microbleeds on head CT or magnetic resonance scan.Exploring the specific clinical hematological markers of VCI to assist in early identification of patient subpopulations,reveal pathogenesis,and help explore possible precise interventions,which may improve prognosis.A large number of clinical and laboratory data have indicated that coagulation/fibrinolytic system,endothelial cell function,inflammation and other indicators closely related to the pathophysiological mechanism of SSVD are expected to become reliable hematological markers.Studies have found that fibrinogen,the terminal protein of the coagulation cascade,as one of the markers of coagulation status,may be related to SSVD.Neuroinflammatory response,malnutrition,immune status imbalance,etc.aggravate white matter damage and participate in promoting the occurrence and development of VCI cognitive impairment.Fibrinogen to prealbumin ratio(FPR)and hemoglobin,albumin,lymphocyte,and platelet score(HALP)were obtained by combining the directly measured fibrinogen,prealbumin,hemoglobin,albumin,lymphocyte,and platelet.FPR and HALP can comprehensively reflect the body’s coagulation/fibrinolysis,inflammation,nutrition,and immune status.Resveratrol(RES)has been shown to improve VCI cognitive function in clinical and basic experiments.Previous studies have confirmed that the stimulator of interferon genes pathway is widely involved in down-regulating inflammatory response and the development of the central nervous system disease.But whether it is involved in the pathophysiology of vascular dementia remains unknown.The role of RES in VD model has not been fully evaluated.There is little study on whether the STING pathway participate in the neuroprotective effect of RES in rats under CCH.In this study,patients with subcortical small vessel disease(SSVD)with white matter lesions as the main feature were selected to explore the relationship between blood markers and white matter lesions and vascular cognitive impairment.Furthermore,VD was simulated in Sprague-Dawley(SD)rats by bilateral common carotid artery occlusion(BCCAO),and the cognitive protective effect and mechanism of RES on rats with vascular dementia was explored,which provided new ideas for clinical research on VD treatment strategies.Part One Correlation between white matter lesions of vascular cognitive impairment and peripheral blood indexes HALP、FPRObjective: To investigate the correlation between white matter lesions of SSVD patients and peripheral blood indexes(FPR and HALP).To gain a deeper understanding of the pathogenesis of SSVD for early diagnosis and treatment of patients with SSVD.Methods: A total of 132 patients with SSVD who were admitted to the department of Neurology of Hebei General Hospital,from July 2021 to July2022,were enrolled according to diagnostic criteria.General clinical data such as gender,age,years of education,past medical history and other general clinical data were recorded.Routine laboratory tests were collected.The peripheral blood indexes were calculated.The cognition of subjects was tested through Montreal cognitive assessment.A cranial MRI scan was performed to collect imaging data.The Fazekas visual semi-quantitative score was applied to assess WMHs grading.According to the grade of WMHs,the study subjects were divided into mild-to-moderate WMHs burden group(1-2 points)and heavy WMHs burden group(3-6 points).The clinical data and peripheral blood indexes were analyzed for differences between the different groups.The correlation between the severity of WMHs burden and hematological indicators and cognitive function was explored.Logistic regression was used to analyze the risk factors for the degree of white matter lesions in SSVD.Results:1.A total of 132 patients with SSVD were included in this study,and these patients were divided into two groups according to the WMHs grading score,63(47.7%)in the mild-to-moderate WMHs burden group and 69(52.3%)in the heavy WMHs burden group.The differences in history of hypertension,years of education,and Mo CA score were statistically significant between the mild-to-moderate WMHs burden group and heavy WMHs burden group(P<0.05).There were no significant differences in sex,age,diabetes history,and coronary atherosclerotic heart disease history between the two groups(P>0.05).2.Compared with the mild-to-moderate WMHs burden group,the serum levels of FPR,TC,LDL-C,VLDL-C and Hcy of heavy WMHs burden group were increased,and the HALP score was reduced,and the difference was statistically significant(P<0.05).While the difference between TG,HDL-C and other indexes was not statistically significant(P>0.05).Correlation analysis showed that the HALP score was negatively correlated with WMHs burden(P<0.05).Hypertension history,FPR、Hcy、TC、VLDL-C and LDL-C was positively correlated with WMHs burden(P<0.05);3.HALP and FPR were all independent influencing factors of the WMHs burden grade.Conclusions:1.Patients with SSVD have cognitive impairment,accompanied by the WMLs.With the increase in the WMHs grade,the cognitive impairment of SSVD patients might be continuously aggravated.2.The FPR of heavy WMHs burden group is increased,and the HALP score is reduced in comparison with the mild-to-moderate WMHs burden group.HALP is negatively correlated with WMHs burden.PFR is positively correlated with WMHs burden.3.FPR is an independent risk factor for heavy WMHs burden of SSVD patients.HALP is an independent protective factor for heavy WMHs burden of SSVD patients.Part Two The protective effect of resveratrol on spatial learning and memory in rats with vascular dementiaObjective: To investigate the effect of RES on spatial learning and memory function of VD rats at fourth and eighth week after the BCCAO surgery.Observe the histological integrity of the rats’ hippocampus.Methods: BCCAO operation on SD rat was adopted to establish the chronic cerebral hypoperfusion of VD.Thirty-six male SD rats(aged 8-10weeks),twelve of them were randomly selected as sham group to receive sham operation and post-operative vehicle.The rest twenty-four rats were randomly selected to underwent BCCAO surgery and subsequently divided into 2VO 4w group(animals underwent 2VO and treated with vehicle for 4weeks after surgery);2VO 8w group(vehicle-treated,sacrificed at 8 weeks after 2VO);2VO+RES 4w group(RES-treated,sacrificed at 4 weeks after2VO);2VO+RES 8w group(RES-treated,sacrificed at 8 weeks after 2VO).The vehicle or RES(20 mg/kg/day)were given to the rats by intraperitoneal injection respectively.Morris water maze was conducted upon the twentythird and the fifty-one day after the operation,include: 1)Place navigation test: The rats were trained four times per day for five consecutive days,and the escape latency time was automatically recorded to reflect the spatial learning ability;2)Spatial probe test: On Day 6,the platform was moved away,the frequency of rats crossed the original platform site,the percentage of the duration that rats stayed in the quadrant of the original platform within120 seconds were recorded to evaluate the spatial memory ability.After Morris water maze test,rats were deeply anesthetized,perfused and fixed by paraformaldehyde.And then the brain slices were embedded in paraffin.HE staining was conducted to observe the hippocampal integrity under optical microscope.Results:1.The escape latency time of the 2VO rats was significantly longer than those of the Sham group on day 2-5(2VO 4w vs.Sham 4w group,2VO 8w vs.Sham 8w group,P<0.05).Moreover,the escape latency of the 2VO 8w rat was longer than those of the 2VO rat sacrificed at 4 weeks(2VO 8w vs.2VO4 w group,P<0.05).While rats in the RES-treated group had a shorter latency than 2VO rats(2VO+RES 8w vs.2VO 8w,2VO+RES 4w vs.2VO 4w group,P<0.05).In the spatial probe tests,rats of 2VO groups spent fewer time in the original platform quadrant and had less platform crossings than those of the sham groups(2VO 4w vs.Sham 4w group,2VO 8w vs.Sham 8w group,P<0.05).However,rats in RES-treated groups spent more time in the original platform quadrant and had a higher frequency of platform crossings(2VO+RES 8w vs.2VO 8w,2VO+RES 4w vs.2VO 4w group,all P<0.05).2.HE stain was used to observe the hippocampal CA1 region of rats.Neurons in hippocampus CA1 region of sham rats were of moderate size,normal shape and microstructure.Whereas neuronal loss and shrinkage and pyknosis of the cytoplasm were observed in the 2VO rats,administration of RES partly alleviated these morphologic changes.Conclusions: RES alleviates the learning and spatial memory deficiency of the VD rats.RES preserves hippocampal integrity.Part Three The protective effect of resveratrol on myelin sheath,and the suppression of microglia and astrocyte proliferation by resveratrol in the white matter of VD ratsObjective: To investigate the effect of RES on myelin sheath and microglia and astrocyte proliferation in CCH rats.Methods: As described above,SD rats were randomly divided into six groups.After the BCCAO or sham operation,RES or vehicle were given by intraperitoneal injection.Rats were sacrificed separately at 4 or 8 weeks after BCCAO.The Luxol fast blue(LFB)staining was performed with paraffinembedded brain tissue sections of corpus callosum.Immunofluorescence staining was conducted to observe the expression of glial fibrillary acidic protein(GFAP)and anti-ionized calcium-binding adaptor molecule 1(Iba-1)in the white matter.Quantitative real-time PCR(q RT-PCR)was applied to measure the m RNA levels of MBP,CD16 and CD206.Protein expression of MBP was tested by western blotting and immunohistochemical staining.Results:1.At 4 weeks after BCCAO,CCH rats showed reduced myelin density in the corpus callosum by LFB stain,also with lower MBP protein and m RNA expression levels compared with the sham groups(all P<0.05).Additionally,loss of myelin density,protein and m RNA expression of MBP were more severe in the 2VO 8w group compared to the 2VO 4w group(2VO 8w vs.2VO 4w group,P<0.05);RES treatment partially restored myelin density,up-regulated percentage of area positive for MBP,elevated the m RNA and protein expression of MBP at 4 and 8 weeks(2VO+RES 8w vs.2VO 8w,2VO+RES 4w vs.2VO 4w group,all P<0.05);2.Compared to the sham groups,the numbers of GFAP+ cells were increased in 2VO groups.By comparison,rats in RES groups exhibited decreased numbers of GFAP+ cells;3.At 4 and 8 weeks post-operation,2VO groups had more Iba-1+ cells vs.Sham groups.Furthermore,2VO groups exhibited higher proportions of microglia with shorter processes.However,RES treatment partially reduced microglial proliferation and activation at both time points;4.Compared to the sham groups,the numbers of cells labeled with Iba-1per area were elevated in the 2VO groups(2VO 4w and 2VO 8w vs.Sham group,P<0.05,respectively).In addition,the model rats sacrificed at 4 weeks after 2VO showed less severe gliosis than those sacrificed at 8 weeks(2VO8w vs.2VO 4w group,P<0.05).Quantitation of Iba-1 positive cells in white matter were partially suppressed by RES treatment at 4 and 8 weeks(2VO+RES 8w vs.2VO 8w,2VO+RES 4w vs.2VO 4w group,all P<0.05).5.A qRT-PCR analysis of the white matter extract showed that the M1 marker CD16 was increased after 2VO but significantly decreased by RES treatment,whereas the M2-type marker(CD206 genes)was increased by RES at 4 and 8 weeks(2VO+RES 8w vs.2VO 8w,2VO+RES 4w vs.2VO 4w group,all P<0.05).Conclusions:1.In the rat model of VD,the myelin density in corpus callosum and the MBP expression in the white matter are decreased in a time-dependent manner,indicating the model of VD is successfully established.The loss of myelin and decreased expression of MBP are accompanied by the decline of spatial learning and memory ability in the rats,suggesting that the loss of myelin in the white matter is related to cognitive decline.RES can protect myelin integrity and alleviate the cognitive impairment.2.In the SD rat model of CCH,the numbers of astrocytes were increased.RES treatment can decrease reactive astrogliosis in white matter,thus relief loss of myelin and promote cognitive recovery.3.In the rat model of CCH,the microglia are proliferated with a greater proportion of M1 type,accompanied by loss of myelin and cognitive decline.However,RES can protect cognitive function,the mechanism may include:suppress the microglia proliferation,promote microglial polarization toward the M2 phenotype and protect myelin sheath.Part Four The mechanism underlying the protective effect of resveratrol on myelin sheath,and the suppression of microglia and astrocyte proliferation by resveratrol in the white matter of VD ratsObjective: To explore the expression of STING/TBK1/IRF3 pathway and related factors in CCH rats and the regulation of RES on this pathway at 4and 8 weeks after BCCAO surgery.Methods: After the BCCAO or sham operation,SD rats were randomly divided into six groups as described above.RES or vehicle were given by intraperitoneal injection.At 4 or 8 weeks after BCCAO,rats in each group were sacrificed to investigate the expression of STING/TBK1/IRF3 pathway and related factors.The total protein of STING,phospho-TBK1,phosphorIRF3,TBK1,IRF3 and the nuclear protein of phosphor-IRF3 was tested by western blotting.Quantitative real-time PCR(q RT-PCR)was utilized to measure the m RNA levels of TNF-α,CXCL10,ICAM-1,VCAM-1 and IL-10.Immunofluorescence double staining was conducted to observe the expression of phosphor-IRF3 in astrocyte,microglia and neuron in CCH rats.Immunohistochemical staining was performed to test MPO expression with paraffinembedded brain tissue sections.Results:1.At 4 and 8 weeks post-BCCAO or sham operation,white matter tissue of rats in 2VO groups showed elevated nucleic acid expression level of these inflammatory cytokines compared to the sham groups(m RNA level of TNF-α,CXCL10,ICAM-1,VCAM-1,all P<0.05).In addition,the m RNA levels of TNF-α,and CXCL10 in the 2VO 8w group were higher than those found in the 2VO 4w group(2VO 8w vs.2VO 4w group,P<0.05).However,treatment with RES partially inhibited this effect until 8 weeks after BCCAO(m RNA levels of TNF-α,CXCL10,ICAM-1,and VCAM-1,all P<0.05).At 4 or 8weeks post-operation,rats in the 2VO groups showed lower m RNA levels of anti-inflammatory cytokines IL-10,while treatment with RES partially reversed m RNA levels of IL-10(P<0.05).At 4 and 8 weeks after BCCAO,compared to the sham groups,the numbers of cells labeled with MPO per area were elevated in the 2VO groups(2VO 4W and 2VO 8W vs.Sham group,P<0.05,respectively).Quantitation of MPO positive cells in white matter were partially suppressed by RES treatment at 4 and 8 weeks(2VO+RES 8w vs.2VO 8w,2VO+RES 4w vs.2VO 4w group,all P<0.05).The MPO protein levels were higher in the white matter sample of 2VO groups than in the corresponding Sham groups at both post-surgery time points(P<0.05),Treatment with RES reduced protein expression of MPO(P<0.05);2.At 4 weeks after BCCAO,the total protein level of STING,phosphoTBK1 and phospho-IRF3 were significantly upregulated in white matter and hippocampus of CCH rats relative to the sham rats(2VO 4w vs.Sham 4w group,all P<0.05).And the m RNA level of IFN-β and IL-1β were significantly upregulated in CCH rats relative to the sham rats(2VO 4w vs.Sham4 w group,all P<0.05).However,treatment with RES partly abrogated this upregulation(2VO+RES 4w vs.2VO 4w group,all P<0.05).At 8 weeks after BCCAO,rats in the CCH group had higher levels of STING/TBK1/IRF3 pathway components compared to the 2VO 4w group(2VO 8w vs.2VO 4w group,all P<0.05).However,treatment with RES decreased their expression at 8 weeks(2VO+RES 8w vs.2VO 8w,all P<0.05).Immunofluorescence showed that,in the sham group,p-IRF3 was detectable in a few neurons,and showed staining almost only in the cytoplasm.Compared to rats in the sham groups,rats under CCH showed an increased number of GFAP+/p-IRF3+,Iba-1+/p-IRF3+ and NEUN/p-IRF3+ double-positive cells at the fourth week,and p-IRF3 was localized in both the cytoplasm and the nucleus.Compared with untreated CCH rats,those treated with RES had less GFAP+/p-IRF3+,Iba-1+/p-IRF3+ and NEUN/p-IRF3+ cells at 4 weeks.The western blotting analyzes showed,CCH could significantly induce the nuclear translocation of p-IRF3 without affecting the total protein expression of IRF3(2VO 4w vs.Sham 4w group,2VO 8w vs.Sham 8w group,P<0.05),whereas treatment of RES counteracted the nuclear translocation of p-IRF3(2VO + RES 8w vs.2VO 8w,2VO + RES 4w vs.2VO 4w group,P<0.05).Conclusions:1.In the CCH rat model,proinflammatory factors including STING,TBK1,IRF3,IFN-β and IL-1β were significantly increased,with the increased astrocyte and microglial reactivity and myelin loss.It suggests that inflammatory response is involved in CCH-induced white matter lesions.The administration of RES significantly suppressed the STING/TBK1/IRF3 pathway and alleviated white matter lesions loss,suggesting that the myelin protection effect of RES is related to the inhibition of STING/TBK1/IRF3 pathway.2.In the CCH rat model,the expressions of TNF-α,CXCL10,ICAM-1and VCAM-1,and the neutrophil infiltration were significantly increased in a time-dependent manner,suggesting the progression of neuroinflammatory responses.The IL-10 was decreased in the untreated-CCH groups,while RES alleviated the down-regulation of the IL-10,decreased neutrophil number and the m RNA expression of TNF-α,CXCL10,ICAM-1 and VCAM-1,suggesting that the protective effects of RES on cognition and myelin is related to suppression of neuroinflammatory responses. |
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