| Intracerebral hemorrhage(ICH) is an common type of stroke. Despite years of research,the treatments for ICH are limited. In animal experiments and clinical practices, we foundthat the brain tissue itself had the ability of hematoma absorption after ICH and the patients’clinical outcomes were positively correlated with the velocity of the hematoma absorption.Therefore, promoting the endogenous hematoma absorption could be a novel treatment forICH.The hematoma components could activate the microglia after ICH. At the early stageof ICH, microglia could clear the hematoma by engulfing the erythrocytes and its dissolvedcomponents. The phagocytic function of the microglia is mainly mediated by the scavengerreceptor CD36, which is participated in the processes of the microglia engulfing theβ-amyloid protein and oxidized low-density lipoprotein(oxLDL), could cause chronicaseptic inflammation, and play an important role in the Alzheimer’s disease andatherosclerosis. However, CD36expression and its role in post-ICH have not beenelucidated.Increasing evidences demonstrated that inflammation was the key factor causingsecondary brain injury following ICH. Studies demonstrated that TLR4participated in thedevelopment of the aseptic inflammation, and had an important role in the central nervoussystem inflammation. In addition, the activation of TLR4signaling produced TNF-α, whichcould downregulate CD36expression in the macrophage and thus affecting its phagocyticfunction. However, whether TLR4signaling in post-ICH could regulate the CD36expression in microglia and thus affecting the phagocytic function of microglia remainsunknown.In the present study, we first utilized the clinical ICH cases to explore the hematomaabsorption and recovery of neural functional in the CD36deficiency patients. Next, we established the in vivo and in vitro ICH model to explore the CD36expression variation andits role in post-ICH and the effect of TLR4signaling on regulating the CD36expression onmicroglia and the phagocytic function. Last, we investigated the effect of the TLR4inhibitor TAK242on regulating the CD36expression and the phagocytic function.Part oneThe impact of CD36-deficency on the hematoma absorption and neurologicdeficit scores of the ICH patientsObjective: To explore the changes of hematoma absorption and corresponding neuralfunction recovery of ICH patients with CD36-deficency, to clarify the function of CD36in the ICH.Methods:199ICH patients’ clinical data and head CT data were collected, and theNIHSS and mRS were conducted. PCR-SSP and Western blot were used to screen theCD36-deficency and its classification identification. Type I CD36-deficency patients werescreening out as CD36-deficency group for the test. Meanwhile, among the CD36normalpatients, patients with the same place and same hemorrhage volume were selected ascontrol group. The hematoma absorption rate and neurological scores of the two groups ofICH patients were compared at multiple time points of ICH development.Results:18CD36-deficency patients (9.0%) were screened and11cases were type ICD36-deficency (5.5%). There is no significant difference in the patients’ basic clinicalinformation, imaging characteristics and biochemical indicators at admittion between theCD36deficiency and CD36normal group. After the same treatment, hematomaabsorption rate on CD36-deficency group was slowed significantly while NIHSS score andmRS score were increased significantly, comparied to the CD36normal patients.Conclusions: The results showed that CD36-deficiency patients’ hematoma absorptionslowed and neurologic deficit scores aggravated, indicating CD36participates in thehematoma absorption, and is associated with the recovery of neural function.Part TwoCD36expression after ICH and its role in the hematoma absorptionObjective: To explore CD36expression and its changes in the brain cells after ICH; tounderstand the changes of hematoma absorption and the phagocytic ability of microgliawith CD36-deficency, and to clarify the role of CD36in the hematoma absorption process. Methods: Firstly, the mice ICH model were established, and Real-time RT-PCR,Western Blot were used to detect the changes of CD36expression after ICH. Secondly,immunofluorescence was used to detect the cell specificity of CD36expresssion after ICH.Thirdly, flow cytometry and immunofluorescence staining were used to observe thephagocytic ability of the CD36-/-microglia on in vitro ICH model. Finally, the changes ofthe hematoma absorption and nerve function defect of the CD36-/-ICH mice were measuredto further confirm the role of CD36in the hematoma absorption after ICH.Results: Real time RT-PCR and Western blot results showed that: after ICH, the CD36expression has increased significantly since day1, peaked at day3, and remained at a highlevel until day7. CD36could express in the neurons, astrocytes and microglias, but mainlyexpressed on the microglias. Microglia can directly engulf RBCs. Phagocytic ability ofCD36-/-microglia was significantly reduced, and the phagocytic ability of wild-typemicroglia was also significantly reduced with the add of CD36antibody. Compared to wildtype ICH mice, hematoma absorption and neural functional recovery of the CD36-/-mousesignificantly slowed, the TNF-α and IL-1β expression were significantly increasedspecifically.Conclusions:1. After ICH, the CD36expression significantly increased, peaked at day3, andmaintained at a high level until day7, which promptes that CD36is involved in thepathological process of ICH. And after ICH, CD36is mainly expressed in microglial cells.2. Phagocytic ability of CD36-/-microglia on red blood cell was reduced, and addingCD36antibody into wild-type microglia had obtained the similar result, suggesting thatCD36plays a very important role in the process of microglia engulfing RBC.3. The results showed hematoma absorption slowed, nerve function defect aggravated,brain water content increased, and the secretion of inflammation factors, such as TNF-α/IL-1β, increased in CD36-/-ICH mouse, which promptes that CD36participates in thehematoma absorption in vivo, and is associated with the prognosis of neural function. Itpromptes that CD36can serve as a new target for the treatment of ICH. Part ThreeTLR4signaling negatively regulates CD36expression and affects hematomaabsorption and mechanism after ICHObjective: To explore the changes of CD36expression on TLR4-/-and MyD88-/-miceafter ICH, study the regulation of CD36expression by TLR4signaling pathways, and testthe effects of TLR4signaling pathways on the hematoma absorption.Methods: CD36expression on TLR4-/-and MyD88-/-mice was measured withWestern Blot after ICH. Hematoma absorption on TLR4-/-and MyD88-/-mice was assessedaccordingly. In the in vitro ICH model, the changes of the CD36expression and phagocyticability of the TLR4-/-and MyD88-/-microglia were measured with Flow Cytometer; and theinfluences of the inflammatory factor on the CD36expression and phagocytic ability ofmicroglia was evaluated with Western Blot and flow cytometry.Results: Compared with wild type mice, the CD36expression on TLR4-/-andMyD88-/-mice was significantly increased, and hematoma absorption was significantlyenhanced after ICH. Compared with wild type microglia, CD36expression of TLR4-/-andMyD88-/-microglia was significantly increased, phagocytic ability was enhanced, and thiseffect can be blocked with CD36antibody. The downstream proinflammatory factor ofTLR4signaling pathways, such as TNF-α and IL-1β, can inhibit microglia CD36expression and weaken its phagocytic ability, among which the TNF-α effect was moreapparent.Conclusion:1. After ICH, increase of CD36expression and hematoma absorption was observed inthe TLR4-/-and MyD88-/-mice, suggesting TLR4signaling pathways is involved in theregulation of CD36expression.2. In the in vitro ICH model, CD36expression was increased on TLR4-/-and MyD88-/-microglia. The RBC engulfing ability of TLR4-/-and MyD88-/-microglia was enhanced andsuch effect could be weakened with CD36antibody. Those results further confirmed thatTLR4signaling pathways are involved in regulation of CD36expression.3. TLR4signaling pathways downstream proinflammatory factor TNF-α, IL-1β caninhibit microglia CD36expression, and weaken the microglial phagocytic ability, and therole of TNF-α seems more important. The results prompted that TLR4signaling pathwaysnegatively regulate the CD36expression via the TNF-α, IL-1β.Part FourTLR4inhibitors promote CD36expression and affect the hematoma absorption after ICHObjective: To assess the influence of TLR4inhibitors TAK242on the CD36expression, microglia phagocytic ability, hematoma absorption after ICH, protective effecton neurons and its possible mechanism.Methods: In the in vitro ICH model, Western Blot and flow cytometry methods wereused to test the influence of TAK242on CD36expression and phagocytic ability ofmicroglia; neurons and microglia transwell were established to test the protective effect ofTAK242on neurons. Real-time RT-PCR were used to measure the influence of TAK242onthe catalase expression. At last, the influence of TAK242on the hematoma absorption afterthe ICH was evaluated in vivo.Results: Enhanced CD36expression and phagocytic ability of microglia werefollowed with TAK242in the in vitro ICH model and such effect could be hindered byinflammatory factor TNF-α, IL-1β. In the neurons and microglia co-culture system,TAK242significantly reduced the number of neuronal apoptosis and necrosis, andincreased microglia catalase expression to reduce the H2O2secretion. In the ICH in vivomodel, TAK242promoted hematoma absorption siginificantly as compared with the controlgroup.Conclusion:1. TAK242increases CD36expression and enhances engulfing RBCs ability ofmicroglia. This upregulation of TAK242is acting through TLR4signaling pathways.2. TAK242increases the microglia catalase expression, reduces the H2O2secretion,and thus playes a protective role of neurons.3. TAK242can not only reduce inflammation injury after ICH but also promote thehematoma absorption, and may become a new drug for the treatment of ICH.This research studied the role of CD36on the ICH hematoma absorption, theregulation of CD36expression and hematoma absorption by the TLR4signaling,demonstrated the regulatory mechanism of ICH hematoma absorption, and provided solidexperimental evidence for a new therapy concept which is to treat ICH actively throughpromoting hematoma absorption. |
