Mechanistic Study Of GJ-4 On Vascular Dementia And The Regulation Of TREM2 On Neuroinflammation

Part 1The therapeutic effects of GJ-4 on vascular dementia rats and the corresponding mechanismsVascular dementia(VD)is a cognitive impairment syndrome caused by a series of cerebrovascular pathological changes.Besides cognitive declines,damage in various brain regions could cause additional symptoms,such as personality changes or language problems,etc.Apart from posing a serious threat to the patient’s physical health,it can also increase their psychological pressure and bring heavy economic burdens to the family and society.Gardenia jasminoides J.Ellis.is a traditional Chinese medicine,which has antioxidative,anti-inflammatory,antibacterial effects,as well as alleviating brain tissue damage caused by ischemia.GJ-4 is extracted from the fruit of Gardenia jasminoides J.Ellis.and it has been proved to alleviate memory deficiency in different Alzheimer’s disease animal models in our previous studies,yet the effects and corresponding mechanisms of GJ-4 on VD has not been fully elucidated and requires further investigation.Therefore,the present experiment was designed to illustrate the therapeutical effects of GJ4 on permanent bilateral common carotid arteries occlusion(2-VO)induced VD rats and investigate the underlying mechanisms through in vivo and in vitro studies.In this study,VD rat model was established through permanent 2-VO,then the rats were intragastrically administrated with different dosages of GJ-4(10,25 and 50 mg/kg),donepezil(5 mg/kg),nimodipine(10 mg/kg)and Ginkgo Biloba leaves(50 mg/kg)for 30 consecutive days.Data from behavioral test showed that GJ-4 50 mg/kg alleviated learning and memory deficits in VD rats by significantly decreasing the latency to find the platform,increasing the number of platform crossings and prolonging the time staying in the target quadrant in Morris water maze test.In the step-down test,GJ-4 50 mg/kg treatment increased the time of rats staying on the platform,indicating that GJ-4 can improve learning and memory abilities in VD rats.Doppler cerebral blood flow result demonstrated that GJ-4 50 mg/kg increased the brain blood flow dramatically.Nissl staining showed that 2-VO resulted nissl bodies distortion in CA1 region of hippocampus,and GJ-4 50 mg/kg markedly alleviated hypoperfusion-induced neural injuries.Apoptosis plays a crucial role in the pathology of VD.Immunohistochemistry staining and Western blot results revealed that GJ-4 could inhibit the expression of Cleaved caspase-3,an important protein involved in apoptosis,indicating that GJ-4 exerted a protective role on neurons by alleviating neuronal apoptosis.Iba-1 staining result demonstrated that GJ-4 may have the effect of inhibiting the activation of microglia in the 2-VO rat model.Misfolded/unfolded proteins accumulation in the lumen of endoplasmic reticulum(ER)may lead to ER dysfunction,initiate a stressed response called endoplasmic reticulum stress(ERS)and activate unfolded protein response(UPR)in order to restore ER protein homeostasis and ER function.Mechanistic studies showed that GJ-4 significantly decreased the expression of ERS-related protein glucoseregulated protein 78(GRP78),and inhibited apoptosis through hindering the activation of PERK/eIF2α/ATF4/CHOP signaling pathway which regulates UPR.For further investigation,oxygen glucose deprivation(OGD)SH-SY5Y cell model was established to mimic the pathological changes of hypoxia neurons.Consistent with the in vivo results,the in vitro data showed that OGD significantly increased ERS related protein GRP78 and activated PERK/eIF2α/ATF4/CHOP signaling pathway,while crocetin,the main metabolite of GJ-4,markedly reduced GPR78 expression and restrained the activation of PERK/eIF2α/ATF4/CHOP signaling pathway.In summary,our study demonstrated that GJ-4 could protect neurons in the brain of 2-VO induced VD rats,alleviate ERS in rats’brain through inhibiting PERK/eIF2α/ATF4/CHOP signaling pathway activation,rescue neurons from apoptosis and improve rats’ learning and memory abilities.Angiogenesis is a process of new vessels growing from existing vessels.Prompting angiogenesis is one of the therapeutic approaches in ischemia induced VD.Wound healing assay and tube formation test were employed for further investigation of the effects crocetin has on human umbilic vein endothelial cells(HUVEC)in this study.The results indicated that crocetin was able to improve HUVEC’s migration and tube formation abilities dramatically.Mechanistic study revealed that crocetin may increase the expression of vascular endothelial growth factor(VEGF)and platelet endothelial adhesion molecule 1(CD31)by activating Sonic Hedgehog signaling pathway,thus prompting angiogenesis.In conclusion,our study showed that GJ-4 improved 2-VO induced brain blood flow reduction,protected neuron from damage in hippocampus,alleviated learning and memory abilities in VD rats.Mechanistic studies demonstrated that inhibiting the activation of UPR regulatory PERK/eIF2α/ATF4/CHOP signaling pathway maybe responsible for the protective role of GJ-4 and its main metabolite crocetin on neurons.Apart from its inhibitory effects on ERS,crocetin could also activate Sonic Hedgehog signaling pathway to prompt migration and tube formation abilities of HUVEC,thus improve HUVEC angiogenesis.Taken together,results of our studies proved that GJ-4 is a promising drug candidate for VD treatment.Part 2Mechanistic study of TREM2 on the regulation of microglial endocytosis in neuroinflammationIn Central nervous system(CNS).triggering receptor expressed on myeloid cells 2(TREM2)mainly locates at the membrane of microglia.TREM2 regulates the survival,proliferation and the ability of migration and phagocytosis of microglia,as well as the inflammatory response.Researches show that neuroinflammation not only participates in but also plays a crucial role in the onset and pathological process of various diseases,including Alzheimer’s disease(AD),Parkinson’s disease(PD),vascular dementia(VD),stroke and traumatic brain injury,etc.Under pathological circumstances,microglia would migrate to lesion spot,clear pathogenic materials,such as β-Amyloid(Aβ)and α-synuclein(α-syn)in order to alleviate neuroinflammatory response.However,the effects and mechanisms of TREM2 on neuroinflammation have not been fully clarified.therefore this study was designed to explore the underlying regulatory mechanism of TREM2 on endocytosis in Lipopolysaccharide(LPS)intraventricular injected mice,OGD-stimulated BV2 microglia and three VD rat models.Results showed that LPS stimulation lowered the expression of TREM2 in microglia.In order to further explore the underlying mechanisms,proteomics was conducted to search for differentially expressed proteins in BV2 cells after TREM2 knockdown by small interference RNA(siRNA)in this study.The results showed that knockdown of TREM2 expression led to decreased expression of clathrin-mediated endocytosis(CME)associated proteins adaptor related protein complex 2 subunit alpha 2(AP2A2),adaptor related protein complex 2 subunit mu 1(AP2M1)and dynamin 2(Dnm2),which was verified by Western blot assay.Co-immunoprecipitation(Co-IP)was then employed to investigate whether TREM2 could interact with these three endocytosis related proteins.The results showed that TREM2 could bind and interact with AP2A2 and Dnm2,respectively,suggesting that TREM2 directly regulated microglial endocytosis.The present study demonstrated that knockdown of TREM2 in BV2 cells markedly inhibited the endocytosis of microglia detected by endocytosis function assay,which validated that TREM2 was closely related to endocytosis.Microglia-mediated neuroinflammation is closely related with endocytosis.In order to investigate the regulatory effects of TREM2 on endocytosis in neuroinflammation,LPS intraventricular injection induced neuroinflammation mice model was established.The results demonstrated that the expression of TREM2 and endocytosis proteins AP2A2,AP2M1 and Dnm2 decreased dramatically in cortex and midbrain of mice stimulated by LPS,meanwhile the expression of inflammatory proteins increased,indicating that LPS damaged the endocytosis function and induced neuroinflammation.Neuroinflammation causes Blood brain barrier(BBB)injuries,and increases the permeability of BBB,thus our study then used Western blot to detect the expression of BBB-related barrier proteins,including Claudin-1 and Occludin.The results showed that LPS significantly reduced the expression of brain barrier proteins.The administration of TREM2 agonist COG 1410 activated TREM2,improved the expression of endocytosis proteins AP2P2,AP2M1 and Dnm2 significantly and decreased the expression of inflammatory proteins,suggesting that TREM2 activation alleviated LPS-induced microglial endocytosis dysfunction and inflammatory response,resulted in improvement in brain barrier function.Neuroinflammation plays a vital role in the pathological changes in VD,thus our study established oxygen glucose deprivation(OGD)model in BV2 cells,aiming to observe the regulatory effects of TREM2 on endocytosis in neuroinflammation under OGD stimulation.The results exhibited that OGD challenge dramatically declined the expression of TREM2 and endocytosis proteins,and increased iNOS and COX-2 expression,indicating that OGD inhibited microglial endocytosis and induced neuroinflammation.COG 1410 administration up-regulated the expression of TREM2,and activation of TREM2 could dramatically improve the endocytosis proteins expression,and alleviated OGD-induced neuroinflammation.Then we explored the effects of TREM2 on endocytosis in neuroinflammation in three VD rat models,including bilateral carotid artery occlusion(2-VO),middle cerebral artery occlusion/reperfusion(MCAO/R),high fat diet+2-VO(HFD+2-VO)rats.Western blot results suggested that in the cortex of all VD rats the expression of TREM2 and endocytosis proteins were reduced dramatically,neuroinflammation was aggravated,and brain barrier was damaged,indicating that TREM2 participated in the pathogenesis of VD by regulating endocytosis.In summary,TREM2 interacted with endocytosis protein AP2A2 and Dnm2 in microglia respectively,mediated microglial endocytosis,and participated in the pathological progress of neuroinflammation related diseases.In neuroinflammation related disease models such as VD,the expression of TREM2 and endocytosis proteins were reduced,while activation of TREM2 increased microglial endocytosis,alleviated inflammatory response and improved brain barrier function.Results above further validated that TREM2 is an important target molecule for neuroinflammation regulation,and highlighted that TREM2 might be a promising target for VD treatment.