| Background: Intracerebral hemorrhage(ICH) is belong to the category of “hemorrhagic stroke” in Traditional Chinese Medicine(TCM). The research of ICH never ceases in modern medical research of TCM. The therapy method for stroke through clearing the hollow viscera is historic.The school, Wang Yong Yan is the representative of a school which recommends that clearing the hollow viscera and reducing phlegm should be used in acute stroke treatment, and the idea has a farreaching influence. The symptom of of constipation even without stool or dry stool of those patients with stroke usually last for 3 to 5 days, even more than ten days. The pathogenesis is deficient root with overdo superficial in the acute phase and overdo superficial takes the main point. Among of them, all of the overdo superficial can highlight, such as the endogenous wind, pathogenic heat, phlegm turbidity and Zang-fu viscera being filled with thing, blood stasis and so on. By this time, the clear substance can not rise and the turbid substance can not descend that induces the confusion of Qi function in rising and falling and blocking of middle burner. Because the middle jiao is the key point of Qi function in rising and falling, so Zang-fu viscera being filled with thing is the most important above of the all. The aperture would not come loose if Zang-fu viscera is filled with thing. The wind would not extinguish if the heat last. So the methods of suppressing hyperactive liver and subsiding yang to calm the adverse-rising energy could not act role in treating stroke because the slow remedy can not meet a urgency. Therefore,bowel-relaxing and purgeing heat is the most appropriate way to treat stroke that can take a drastic measure to deal with a situation. Dachengqi tang is used the most commonly among the way of clearing the hollow viscera and its role and mechanism of treatment ICH needs further studies.Inflammation and oxidative stress are the most important mechanism after ICH. Among them, the microglia was activated and acquired phagocytosis to remove the hematoma and necrotic debris procreanted by brain tissue and erythrocytes. Activated microglia can express and release of inflammatory factors, thus aggravate brain damage. The controversy continued whether microglia’s activation should be inhibited or promoted. Furthermore, how to effectively judge the degree of microglia being activated and what about the shape and function, all of these are worth studying. The Nrf2/Src/MAPKs signal pathway is actived in inflammation and oxidative stress after ICH. Whether and how do Da Cheng Qi Tang and emodin effect on the signal pathway? It would reveal the mechanism of action of Da Cheng Qi Tang and emodin.Aim: Study on the role, function and mechanism of signal pathway of dachengqi tang and emodin on ICH model rats induced by collagenase VII. Meanwhile, study on the action of dachengqi tang and emodin on microglia, discuss the characteristic and rule of actived microglia after ICH.Methods: In this study, the SD rats were divided into control, model, Dachengqi tang and emodin groups randomly. The collagenase VII was used to injecte in the left caudate putamen(CPU) of rats to copy the model of ICH except the control group which injected with normal saline. The dachengqi tang and emodin groups were gavaged with dachengqi tang(1 g /100 g weight) and emodin(10mg/100 g weight) respectively while the control and modle groups gavaged with normal saline.The general condition and change of weight were used to assess the entirety disease state and degree of recovering. 2,3,5- triphenyl four azole nitrogen chloride(TTC)staining and magnetic resonance imaging(MRI) were used to observe and estimate the morphological change of rats brain after the collagenase VII was used for 3 days. Then the balance beam test and elevated body swing test were used to detect and evaluated the change of myodynamia and neurologic impairment, as well as MRI was used to observe the volume of hematoma to evaluate the scope of brain injured after ICH. Prussian-blue staining was used to detect the phagocytesis and deposition of microglia to iron to evaluate the pathologic change and recovery of ICH model rats.Histochemistry staining of ionized calcium binding adaptor molecule 1(Iba1) was used to detect the activation and regular way of microglia. Histochemistry and double immunofluorescence staining of interleukin-1β(IL-1β), IL-10, TNFα were used to detect and analyze the types and levels of cytokines secreted by actived microglia. Then western blot was used to detect and analyse the Nrf2/Src signal pathway and its downstream mitogen-activated protein kinases(MAPKs) such as extracelluar signal-regulated kinase(ERK), jun N-terminal kinase(JNK) and P38. Meanwhile, the expression level of promote inflammatory factor such as IL-1β and TNF-α and anti-inflammatory factor such as Arg1 and IL-10 were also detected and analysed by western blot.Results: We observed that the general station of rats was bad and weight decreased when collagenase VII was injected in left CPU of rats for 3 days. TTC staining showed the construction of left locality brain tissue damaged obviously. In beam balance test, collagenase VII injected rats had more drop times on the 80 cm long beam in 2 minites than control rats and they often fell down from the right side. In elevated body swing test, collagenase VII injected rats showed more left-biased swings than control rats at 1-day, 3-day and 7-day after injection. These tests showed obvious decreased muscle strength of the right limbs in collagenase VII injected rats. The dated of MRI showed there were high signal region with the irregular shape in the left CPU that meaned there were hemorrhage region there. The hemorrhagic focus in CPU was clearly showed by brain MRI test at 3-day after injection. These data showed that collagenase VII injection successfully duplicated CPU hemorrhage in rats.The neurologic impairment was improved and hemorrhage region was reduced after dachengqi tang and emodin being used to treated ICH rats at 28 days. Prussian-blue staining showed that dachengqi tang and emodin could shallow the coulor which indicated they could accelerate the elimination of near iron. To verify the cells around the hemorrhagic focus, we stained the brain slices of collagenase VII injected rats at 1 day after operation with Iba-1(marker of microglia) and GFAP(marker of astrocyte) by immunohistochemistry. Both microglia and astrocyte were actived in the collagenase VII injected CPU. However, microglia, but not astrocyte, was observed more close to the hemorrhagic focus by double-label immunofluorescence staining, indicating the important role of microglia in the pathological changes following ICH. Microglia is sensitive to brain injury, altering their morphology and phenotype to adopt a so-called activated state in response to brain insults. Activated microglia could migrate to the insult focus, swallow the dying cells and debris, and release some cytokines to maintain the homeostasis of microenvironment. In brain slices taken at 1-, 3-, 7-day after operation, we observed the obviously increased microglia in collagenase VII-injected CPU. According to the distance to the center of hemorrhagic focus, we selected four areas to analysis the morphologic feature of activated microglia, and selected the contralateral CPU as control area. In region I, the number of microglial processes was decreased compared with those in control area. The processes were still radial distributed in region I. In region II, III, and IV, the decreasing of microglial processes was observed. Especially in region IV, where microglia had the fewest processes number, most ball-like microglia even lost their processes. Region III had the largest density of microglia, and the concentrated microglia arrayed around the hemorrhagic focus. In region II and III the processes were not radial distributed, they have the processes mainly in the side towards the hemorrhagic focus. To evaluate the process directivity of a microglial cell, the cell body was set as origin, and the Y axis was set pointing to the hemorrhagic focus. The process directivity of a microglial cell was calculated as that the processes number in quadrant I and IV deduct the processes number in quadrant II and III, then the difference was divided by the total processes number. We found the microglia in region II and III had higher directivity towards the hemorrhagic focus. These data showed that the activeted microglia in different region have different morphological characteristics. Summarily, in region I the radial distributed processes of microglia have a decreased processes number. Microglia in region II and III had their processes mainly towards the hemorrhagic focus. Region III had the largest density of microglia, and the microglia compactly arrayed around the hemorrhagic focus like a barrier between injuried and uninjuried parts. Ball-like microglia in region IV had the fewest number of processes. The acreage of microglia cell body was negative correlation to the distance of central zone of hemorrhage. The method of area analysis of microglia could not effectively analyse the microglia in bleeding area and the surrounding area. Then we designed a new method of analysis, named microglia activation factor analysis method(MAF), which could tell activation degree in a quantitative way. Using this method, we found within 400 μm from the center of the ICH, microglia activation degree was negative correlation to the distance of central zone of hemorrhage.After ICH, the form and function of microglia were changed, which was closely related to the spatial location of microglial cells. Activated microglia could phagocytoses erythrocytes effusion, including necrotic erythrocytes and brain tissue pieces, which resulted in the deposion of iron in cell membrane, cytoplasm and/or nucleus. In region IV, we found lots of spots around ball-like microglia, indicated the microglia may phagocytosis something. To understand the possible function of microglia in region IV, Prussian-blue staining, which is also named hemosiderin staining, was used. After the erythrocytes being phagocytosised into macrophage/microglia, hemoglobin in erythrocytes will be decomposed into hematoidin and hemosiderin by lysosomal enzyme. Hemosiderin presents blue after Prussian-blue staining. In this research, we found blue-staining region mainly located around hemorrhagic focus from the brain slices taken at 3-day, 7-day, 14-day, and 28-day after collagenase VII injection. Brain slices taken at 7-day, 14-day, and 28-day showed stronger blue-staining than 3-day brain slices. After double staining the brain slices with Iba-1, a marker of microglia, and Prussian-blue staining, we found most microglia in region IV were Prussian-blue positive. These data indicate that ball-like microglia in region IV has strong ability of phagocytosis and they play important role in the cleaning of hemorrhage.By histochemistry, we discovered the actived microglia was M1 type and mainly secreted interleukin-1β(IL-1β) but hardly not tumor necrosis factor(TNF-α) at 3 days after ICH, this station could continued about 7 days. After 7 days, the M2 type of actived microglia was dominant.By western blot, we observed that dachengqi tang and emodin could reduce the expression of IL-1β, but not TNF-α in CPU. At the same time, dachengqi tang could up-regulated the expression of arginine 1(Arg 1) and IL-10, while emodin only could up-regulated the level of IL-10. In hippocampus, dachengqi tang and emodin had no action on IL-1β and TNF-α, but both could increase the level of Arg 1. Furthermore, dachengqi tang also could increased the expression of IL-10 compared with the model group. In CPU, dachengqi tang also could up-regulated the level of phosphorylated extracelluar signal-regulated kinase(P-ERK), downregulated the level of phosphorylated jun N-terminal kinase(P-JNK), but had no action on the level of phosphorylated P38(P-P38). As for emodin, it could increse the level of P-ERK, P-JNK and P-P38. Both dachengqi tang and emodin could reduced the level of nuclear factor erythroid-2 related factor 2(Nrf2) and/or Src. All results were based on datd of 14-day after collagenase VII injection.Conclusion: Our findings suggest that, 1.treatment with dachengqi tang and emodin, can improve neurologic impairment, reduce volume of hemorrhage, recover the pathological changes induced by deposition of iron, so as to shorten the process of pathological of ICH. 2.the treatment effect involves increasing the level of Arg1、IL-10, down-regulating the level of IL-1β through inhibiting JNK in CPU, while down-regulating the level of TNF-α through inhibiting P38 in hippocampus. These data show it that the role of dachengqi tang and emodin realize through Nrf2/Src signaling pathway. 3.activated microglia possesses morphological and functional plasticity and they match each other in the process of ICH. 4.MAF can judge the degree of miroglia activation accurately through the form of quantitative way. |
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