| In recent, there has been growing concerning about treatment of acute stroke which has implications for prognosis and rehabil itat ion. In Tranditional Chinese medicine (TCM), "reinforcing the healthy qi" and "eliminating the pathogenic factors" are the main methods of treatment throughout the pathological process of stroke. Current trends in treatment of acute stroke was with an emphasis on "eliminating the pathogenic factors", whereas through summarizing the clinical experience, "reinforcing the healthy qi" were found exerting significant effect to improve prognosis and rehabilitation. With the integrating disease and syndrome pattern, this study is to explore positive function of "reinforcing the healthy qi " treatment in acute phase on improving prognosis and rehabilitation from the perspective of "combination with kindey and brain" in order to provide the biomark of evaluting the effecacy of acute stroke. Theory research:The theory about "combination with kindey and brain" was screened and catagoried with respects to organizational structure, Physiological function, Pathogenesis and application of herbs from 650 ancient medical books involved in an e-book archive. The kidney stores the essence, essence produces marrow, marrow connects to brain, and brain is the convergence of marrow, the brain and kidney have close relationship. Organizational structure and physiological function not only composes an important basis of interaction on the pathogenesis, but also provides the theoretical basis of clinical treatment for acute stroke. Animal experiment:Objective:To investigate dynamic level changes of ACTH, CRH and COR of MCAO rats at different time point, expression levels of ACTH, CRH, COR were detected by ELISA at 6h and 48h after cerebral ischemia. To determine if Dihuangyinzi decoction interferes with the HPA axis, apoptosis process and the expression of HSP70 in MCAO rats and to explore the role of Dihuangyinzi decoction in neuroprotection effect and improving the disturbed condition of HPA axis, the morphological changes of neural cell and apoptosis in cerebral histiocyte were detected by staining brains slices with hematoxylin-eosin(HE) and imunohistochemical (IHC) method. In summary, this study was with the aim of revealing neuroprotection mechanism and basis theory with application of reinforcing kidney herbs in acute phase of stroke.Method:Male Sprague-Dawley (SD) rats of body weight 280±10g were used for this test.Part one:48 SD rats were randomly divided into 3 groups:normal group, sham operatedgroup and MCAO model group, each group was 16. MCAO was induced by inserting a nylon monofilament through the external carotid artery into theintracranial circulation to occlude the middle cerebral artery at its origin. SD rats were killed 6 or 48h after cerebral ischemia. The expression levels of ACTH, CRH, COR were detected by ELISA at 6h and 48h after cerebral ischemia.Furthemore, the brain specimens were fixed in paraformaldehyde, embedded in paraffin, and then cut into Sagittal slices of 4μm thickness.To detect the morphological changes of neural cell and apoptosis in cerebr al histiocytes, brain slices were staind with hematoxylin-eosin (HE) and Ini-munohistochemical(IHC) method, respectively.Part two:30 SD rats were randomly divided into 3 groups:MCAO model group, herb formula group,and sham operated group,each group was 10. For drug tre atment, herb formula group were given Dihuangyinzi decoction from 2days to 8days after ischemia (1ml/kg body mass) by intragastric administration met hod, whereas the MCAO model group and sham operated group received equivale nt volumes of distilled water. SD rats were killed 1h after the last intrag astric administration. The expression levels of ACTH, CRH, COR were detecte d by ELISA. Furthemore, the brain specimens were fixed in paraformaldehyde, embedded in paraffin, and then cut into Sagittal slices of 4μm thickness. To detect the morphological changes of neural cell, apoptosis in cerebral histiocytes and the expression of HSP70, brain slices were staind with hema toxylin-eosin(HE) and imunohistochemical (IHC) method, respectively.Result:1. Expression levels of ACTH, CRH and COR in serumThe levels of ACTH, CRH and COR at 6h after ischemia were significantly lower in MCAO model group and sham operated group than in normal group(P<0.05); the levels of ACTH, COR were significantly greater in MCAO model group at 48h after ischemia than that in normal group and MCAO model group at 6h after ischemia(P<0.05); the level of CRH at 48h after ischemia in MCAO model group was significantly lower in MCAO model group at 6h after ischemia and in normal group (P<0.05).2. Apoptosis in cerebral histiocytes of MCAO rats at different time point.TUNEL-positive cerebral histiocytes were found by light microscope in MCAO model group at 6h and 48h after ischemia specimens (vs normal group and sham operated group, P<0.01), however, there was no significant difference between MCAO model group at 6h after ischemia and MCAO model group at 48h after ischemia. 3 Dihuangyinzi decoction interferes with the HPA axis serum reaction of MCAO At 8days after ischemia, the levels of ACTH, CRH were significantly lower in MCAO model group than in normal group (P<0.05), the level of COR in MCAO model group were significantly higher than that in normal group; The level of COR was significantly lower in herb formula group than in normal and MCAO model group; The levels of ACTH, CRH were significantly higher in herb formula group than in MCAO model group, the levels of ACTH, CRH were lower in herb formula group than in normal group withou significant difference.A reverse change was observed between the level of ACTH, CRH and the level of COR.4. Dihuangyinzi decoction interferes with apoptosis in cerebral histiocytes of MCAO rats.At 7days after intragastric administration, TUNEL-positive cerebral histiocytes were found in MCAO model group and herb formula group (vs normal group, P<0.01); The level of apoptosis in cerebral histiocytes was significantly greater in MCAO model group than in herb formula group (P<0.01).5. Correlation analysis between apoptosis in cerebral histiocytes and HPA axis serum reaction of MCAO rats.In MCAO model group, there was significantly negative correlation between the level of ACTH and apoptosis in cerebral histiocytes by TUNEL staining, while there were no correlations between the levels of CRH, COR and the apoptosis in cerebral histiocytes. In herb formula group, there were no correlations between the levels of ACTH,CRH, COR and the apoptosis in cerebral histiocytes.6. Dihuangyinzi decoction interferes with the expression of HSP70 in cerebral tissu of MCAO rats.The expression of HSP70 were found in MCAO model group and herb formula group (vs normal group, P<0.05); The expression of HSP70 was significantly greater in herb formula group than in MCAO model group (P<0.05).7. Correlation analysis between the expression of HSP70 in cerebral tissu and HPA axis serum reaction of MCAO rats.In MCAO model group, there was significantly positive correlation between the level of COR and the expression of HSP70, while there were no correlations between the levels of CRH, ACTH and the expression of HSP70. In herb formula group, there were no correlations between the levels of ACTH,CRH, COR and the expression of HSP70 in cerebral tissue.Conclusion:1. At 6h after ischemia, HPA axis serum reaction was in an inhibited state. At 48h after ischemia, HPA axis serum reaction was in an obviously disturbed condition which might lead to more complicated syptoms and higher mortality. 2. Dihuangyinzi decoction can improve the holistic state of MCAO rats and prevent from weight loss.3. Dihuangyinzi decoction can inhibit the release of COR by increasing the level of CRH, which can improve the disturbed condition of HPA axis serum reaction.4. Dihuangyinzi decoction can obviously inhibit apoptosis in cerebral histiocytes, which may have implication for improving the disturbed condition of HPA axis serum reaction5. Dihuangyinzi decoction can exert neuroprotection effect by enhancing the expression of HSP70 in the ischemia cerebral tissue.Clinical reseach:Under the guidance of "integrating disease and syndrome" research model, this study revealed the correlations between TCM syndrome and biomarker in acute phase of ischemic stroke. All the cases of this study have been chosen from National Point Foundation Research Development Plan (973 plan)---Ischemic Stroke Diagnosis Combining Symptoms and Signs of Disease and Effect Evaluation System Research. (No.2003CB517102). In this study, to explore the relationship between biomarker and TCM syndrome and to develop the diagnosic standards for ischemic stroke under the "integrating disease and syndrome" pattern, " Ischemic Stroke TCM Syndrome Factor Diagnostic Scale" (ISTSFDS) was collected for Syndrome diagnosis and serum samples were collected for detecting the levels of ACTH, COR and HSP70. In summary, this study may provide the theoretical basis of TCM neurology. |
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