| This study is divided into 3 parts:1. CD163 expression after ICH and its role in the hematoma absorption and inflammation This part of the study by observing the changes of CD163 expression in perihematoma tissue in mouse intracerebral hemorrhage model, the relationship between CD163 and TLR4 signaling pathway has been explored, thus the role of CD163 in absorption of hematoma and inflammation after cerebral hemorrhage was clear. Intracerebral hemorrhage model was established in mouse and the changes of CD163 expression have been observed after intracerebral hemorrhage by real-time PCR and western blot. Using immunofluorescence method, we observed the expression of CD163 in mouse brain microglial cells. Expression of TLR4 signal pathway and inflammatory cytokines were detected in perihematoma tissue of mouse by Western Blot. The results showed that compared with the Sham group, CD163 expression was significantly higher in the 1st day,3rd day to peak and 7th day still higher. CD163 expression mainly in microglia, and the largest number of positive cells was decected on the 3rd day. TLR4, TRIF, My D88 were increased on 1st day, and reached a peak, then gradually decreased. However, NK-κBp65 appeared increasing on 1st day and 3rd day to peak. Both of TNF-α, IL-10 increased on 1st day, and reached a peak then gradually decreased, 7th day still high. Therefore, after intracerebral hemorrhage, CD163 is mainly expressed on microglial cells involved in the pathogenesis of cerebral hemorrhage. TLR4 signaling pathways involved in the inflammatory reaction after cerebral hemorrhage, may regulate the CD163-mediated absorption of hematoma. CD163 expression could inhibit pro-inflammatory, and play important rolr in anti-inflammatory effects.2. To investigate the interaction between CD163 and TLR4 signaling pathway after intracerebral hemorrhage in vitroIn this part of the study we used BV2 cells to establish intracerebral hemorrhage model in vitro. By transfection si RNA-CD163 and TLR4 antagonist TAK242 intervention to BV2 cells, the interaction between CD163 and TLR4 pathway was observed. Detection capability of hemoglobin phagocytosis, the changes protein of expression including CD163,TLR4 and other contents have been observed by western blot. Expression of cytokines were detected by ELISA. Transfected microglial cells phagocytic capacity of hemoglobin decreased(P <0.05), the amount of CD163 expression reduced(P <0.01), however,TLR4, My D88,TRIF expression was significantly increased(P <0.05). Whereas blocking TLR4 pathway, the absorbance of hemoglobin decreased, that microglia enhanced phagocytic ability of hemoglobin(P <0.01). There was an increase in CD163 expression, but TLR4, My D88, TRIF expression levels decreased(P <0.05). Compared with Sham group, the expression of CD163 in TNF-α group and IL-1β group were significantly decreased(P <0.05), which in TNF-α group decreased more significantly(P <0.01). On the contrary, the expression of CD163 in IL-10 group was significantly higher than that in Sham group(P <0.05). We believe that the participation of intracerebral hemorrhage after CD163 absorption, TLR4 signaling pathway were primarily negative regulatory role to CD163 by TNF-α, IL-1β. TLR4 antagonist TAK242 may become new drug treatment of cerebral hemorrhage.3.CD163 promotes hematoma absorption and improves neurological functions in patients with intracerebral hemorrhage Former studies indicate an association between hematoma absorption and prognosis for people with intracerebral hemorrhage, however, the mechanism of hematoma absorption is unclear. CD163 plays an important role in the metabolism of hemoglobin, and is considered to be a blood biomarker of many diseases. There are no reports about serum CD163 level in intracerebral hemorrhage patients. This study aims to examine the correlation between CD163 and the hematoma absorption and corresponding neurological function recovery of intracerebral hemorrhage patients, and to clarify the function of CD163 in the intracerebral hemorrhage. We measured serum Soluble CD163 levels in intracerebral hemorrhage patients. According to the average of CD163 levels, all the patients were divided into Low level(L) group and high level(H) group. Significantly difference in the concentration of soluble CD163 was observed between participants of the two group(P<0.01). After the same treatment, hematoma absorption rate in the L group was significantly slower than that in the H group. While the National Institutes of Health Stroke Scale scores and modified Rankin Scale scores in the L group were increased significantly, compared to the H group(P<0.05). CD163 functions in promoting hematoma absorption and prognosis in patients with intracerebral hemorrhage was found. We also identify possible therapeutic options for the treatment of intracerebral hemorrhage. |
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