Containing The Protective Effect And Mechanism Study Of The Rat Hemorrhagic Brain Injury Without Cinnabar, Realgar Angongniuhuang Pill

Intra-cerebral hemorrhage (ICH) is also called non-wounded cerebral hemorrhage. It belongs to one of the category of stoke. Among all the stroke patients, the number of ICH patients is going up to 20%³0%, and the hospital mortality has reached 43%⁵1%. Nowadays, the onset age of ICH tends to be younger, therefore, more and more attention is attracted to the research on prevention and therapy of ICH. Generally speaking, ICH is caused by the attack of both wind and fire, and the dampness is more one bad factor.With regard to the therapy on ICH, Traditional Chinese Medicine (TCM) has its own unique curative effect. The basic principle of TCM is to apply different prescriotions to treat different patients, that is to say, disease and syndrome are taken into account all together when facing different patient.Angongniuhuang pill (AGNHW), the fomous Chinese medicine has remarkable effects of removing heat and cleaning toxin, so it has good curative effect on ICH. But for a long time, it is argued whether it is necessary to add Cinnabaris and Realgar into the medicine because of their huge toxicity. Therefore, We design a series of experiments so as to clarify the difference of AGNHW with Cinnabaris and Realgar and AGNHW without Cinnabaris and Realgar (QZX-AGNHW). And the chief results are described as follows.PART ONE : Protective effects and its mechanism on neurons in rats with ICH of AGNHW with or wihtout Cinnabaris and Realgar1 Protective effects and its mechanism on neurons in rats with ICH of AGNHW containing Cinnabaris and Realgar or notObjective : To study the effects of AGNHW on nerve symptoms, cerebral index cerebral edema, antioxidation capacity, blood rheology and morphological changes. At the same time, the effect of QZX-AGNHW and the difference between the two kinds of medicines are observed. Methods: Collagenase-Induced intra-cerebral hemorrhage model in rat caudate nucleus was applied. Nerve symptoms were scored at the fourth day after the operation, moreover, the other factors such as the cerebral index , the content of water , antioxidation capacity, blood rheology and morphological changes were tested at the same time. Results:(1)In the vehicle group, nerve symptoms, content of water , cerebral index,content of MDA, activity of LDH increased greatly. (2)The dosage of 200mg/kg, 400mg/kg of AGNHW can improve significantly the symdrome (vs vehicle p<0.01or p<0.05) . In the vehicle group, the activity of SOD, erythrocyte deformability decreased greatly, on the contrary, the dosage of 200mg/kg, 400mg/kg of AGNHW increased these targets significantly ,( vs vehicle, p<0.01or p<0.05) . (3)Compared with AGNHW, the function of improving nerve symptoms and decreasing cerebral index of QZX- AGNHW was weakened. Conclusion:(1)AGNHW has protective effect on ICH, and it can improve nerve symptoms and content of water, also, decrease cerebral index, content of MDA and the activity of LDH. (2) The function of improving nerve symptoms and decreasing cerebral index of QZX-AGNHW was weakened.2. Effects on the expressions of apoptosis - related proteinas BBB associated protein in rats with ICH of AGNHW containing Cinnabaris and Realgar or not Objective: To approach the effect of AGNHW on the apoptosis and apoptosis- related proteinas BBB associated with protein expressions. and compare the difference of the effects of AGNHW and QZX-AGNHW. Methods: Expressions of apoptisis (TUNEL), apoptisis -associated protein (Bcl-2,Bax)BBB associated protein (MMP9, AQP4)were observed by immunohistochcmistry of AGNHW on perihematomal isscue in rats with ICH. Results:(1)In the vehicle group, The expression of TUNEL, Bax, MMP9, AQP4 were increased, and the expressions of Bcl-2 deceased. (2) The dosage of 400mg/kg of AGNHW can decreased the expression of TUNEL, Bax, MMP9 (vs vehicle p<0.05). On the other hand, it can increase the espression of Bcl-2 (Vs vehicle, p<0.05). The dosage of 200mg/kg ,400mg/kg of AGNHW can decrease the expression of AQP4(Vs vehicle p<0.05, or p<0.01, respectively).(3)The dosage of 160mg/kg, 320mg/kg of QZX-AGNHW decrese the expression of MMP9, AQP4 (Vs vehicle, p<0.05 or p<0.05). Conclusion:(1)AGNHW protect neurons by affecting anti-apoptosis and related BBB permeability. (2)The function of related BBB permeability of QZX- AGNHW was similar to AGNHW, while the influence of anti-apoptosis decreased obviously.3.Effects on MAPK signal transduction pathway in rats with ICH of AGNHW containing Cinnabaris and Realgar or notObjective: To observe the effect of AGNHW and QZX-AGNHW on MAPK signal transduction pathway in rats with ICH, and compare their difference. Methods: Expression of the MAPK signal transduction pathway associated protein PKC,c-fos and c-jun was observed by immunohistochcmical. Expression of the MAPK signal transduction pathway family ERK,P38mRNA was observed by real time RT-PCR. Results: (1)The expression of PKC,c-fos,c-jun inceased after ICH injury, and the expressions of ERKmRNA deceased. However, the expression of P38 mRNA increased (Vs vehicle, p<0.01). (2) The dosage of 200mg/kg, 400mg/kg of AGNHW reduced the expression of c-fos, c-jun (Vs vehicle, p<0.01, p<0.05), The dosage of 400mg/kg of AGNHW reduced the expression of P38mRNA and PKC, and improve the expression of ERKmRNA(Vs vehicle, p<0.05). (3) The dosage of 160mg/kg, 320 mg/kg of QZX-AGNHW decrease c-fos expression(Vs vehicle, p<0.05 or p<0.01),and the dosage of 160mg/kg decrease PKC expression(Vs vehicle, p<0.05), the dosage of 320mg/kg decrease the expression of c-jun , P38mRNA, (Vs vehicle, p<0.01),and increase ERKmRNA expression (Vs vehicle, p<0.01). Conclusions: (1)Protective effects of AGNHW on neurons in rats with ICH was observed by influence activity of MAPK signal transduction pathway.(2)The innfluence of QZX-AGNHW on MAPK signal transduction path-way was similar to AGNHW, but its function of decreasing expression of P38mRNA was weakened. Part two: Protective effects and its mechanism on primary cultured cortex neuron of AGNHW containing Cinnabaris and Realgar or not with serum or CSF containing drugProtective effect of serum or CSF containing drug was observed on oxygen-glucose deprivation or GLU inducing cultured cortex neuron model. The influence of serum or CSF containing drug on MMP and [Ca²⁺]i of GLU induced cultured cortex neuron model. Contents of determination Cinnaba and Realgar in serum or CSF containing drug were determined. 4 Protective effects and its mechanism on primary cultured cortex neuron of AGNHW containing Cinnabaris and Realgar or not with serum or CSF containing drugObjective: To compare the protective effects of AGNHW and QZX-AGNHW with serum or CSF containing drug. Methods: Using cell damage models induced by oxygen/glucose deprivation and GLU,the serum and cerebrospinal fluid pharmacological methods were determined, in order to observe the function of cinnabar and realgar. Results: (1) The damage of oxygen-glucose deprivation increased the leakage rate of LDH. (2)observe morphocytological chages and measure the LDH leak rate , cytoactive , intracellular concentration of MDA and SOD. In the cells injured by the oxygen/glucose deprivation,LDH leak rate was significantly increased, while the cytoactivty was inhibited. These changes were blocked effectively by serum and cerebrospinal fluid containing AGNHW, especialy by the dose of 800mg/kg,and the cerebrospinal fluid with the additive proportion of 20% is better than the same dose. The result indicated that the effection of AGNHW is better than that of QZX-AGNHW. After cells were injured by GLU,LDH leak rate was also significantly increased,meanwhile, the cytoactive was inhibited,and morphocytologic changes were observed obviously. AGNHW had the significant protective actions when the dose reached 640mg/kg,and the cerebro spinal fluid with the additive proportion of 20% was better using the same dose. Conclusion: AGNHW has significant effects of protecting cells.5 The effects on mitochondria membrane potential and intracellular Ca²⁺ concentration of rat cortical neurons injured by GLU of AGNHW and QZX-AGNHWObjective: To observe the effects of AGNHW and QZX-AGNHW on mitochondria membrane potential and intracellular Ca²⁺ concentration of rat cortical neurons injured by GLU. Methods: Using the method of flow cytometry(FCM),mitochondria membrane potential and intracellular Ca²⁺ concentration of rat cortical neurons were measured, then the effects of AGNHW and QZX-AGNHW were compared on the basis of the above data. Results: In rats whose cortical neurons were injured by GLU,mitochondria membrane potential was observed to decrease,and the concentration of intracellular Ca²⁺ raised. The value of mitochondria membrane potential could be increased by AGNHW and QZX-AGNHW containing serum and cerebrospinal fluid. While the concentration of intracellular Ca²⁺ reduced evidently when acted by AGNHW containing cerebrospinal fluid and QZX-AGNHW containing serum and cerebrospinal fluid. Conclusion: AGNHW and QZX-AGNHW had the similar effects, which produced the protective effects on injuryed neurons through stabilizing mitochondria membrane potential and blocking calcium overload.6 Determination of Concentration of cinnabar and realgar in serum and CSF of AGNHW and QZX-AGNHWObjective: To determine whether As and Hg contained in cinnabar and realgar were absorbed into blood or cerebrospinal fluid (CSF) through BBB. And further to analyze if cinnabar and realgar play an important role in the protective effect of AGNHW. Methods: Inductively Coupled Plasma-mass was used to determine the concentration of As and Hg in serum and cerebrospinal fluid . Results:( 1) As in AGNHW could be tested in blood and might be absorbed into the CSF through BBB. The content of As in serum is 5.26 times that of As in CSF.(2) Trace As could be tested in serum and CSF of normal control group and QZX-AGNHW group.(3) The content of Hg was very near the limit of detection of instrument, so it could not be detected exactly. that is to say, the content of Hg need more accurate methods to be detected. Conclusion: Both Serum and CSF Contaning Drug of AGNHW contained As and Hg,the difference of neuro protective effect of AGNHW and QZX-AGNHW had relationship with As and Hg.In summary, AGNHW alleviated the neurologic symptoms of rats after ICH; amelioration morphology changes induced by ICH; reduce cerebral index , improve erythrocyte deformability, all these results show its satisfactory protective effect to experimental ICH. The main mechanism of effect may as follows: It can relieve the injury induced by the toxic product such as excitatory amino acids(EAA), MDA and free redical. In addition, partially inhibit calcium overload induced by EAA, stabilize the mitochondria membrane potential. It has the function of anti-apoptosis by increasing the express of Bcl-2,and decreasing the express of Bax. It can surpression the expression of c-fos,c-jun,PKC,elevate the mRNA expression of ERK and decrease the mRNA expression of P38.their specific receptor. It can surpression the expression of AQP4,MMP9.Compaired with AGNHW , the function of QZX- AGNHW of increasing nerve symptoms, decreasing cerebral index was weakened .And the function of anti-apoptosis of QZX- AGNHW is not notable.In this experiment ,we studied the protective effect of AGNHW and QZX- AGNHW to ICH rats.1 In this study, the protective function of AGNHW and QZX-AGNHW on rats injured with ICH was analyzed. Accordingly entirety pharmacodynamics,serum phamacology and CSF phamacology , the effective differences of the two kinds of medicines were compared. The results show that cinnabar and realgar play an important role in the total prescription.2 Based on the experiments of the mechanism analysis, AGNHW and QZX-AGNHW have the similar effects, which produce the protective effects on injuryed neurons through anti-apoptosis and blocking calcium overload. 3 For the first time, it is reported that the effection of AGNHW and QZX-AGNHW on the pathway of signal transconduction of MAPK.