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Objective:To observe the effect and mechanism of Tanhuo Prescription on regulating the activation of M1 microglia and alleviating brain tissue injury in rats with cerebral ischemia.Methods:Male SD rats were divided into sham-operation group, model group, Tanhuo Prescription high-(3.68 g/kg), medium-(1.84 g/kg), low-dosage(0.92 g/kg) groups, and ginaton group (0.06 g/kg) using random number table method. Except for the sham-operation group, the other groups established cerebral ischemia rat models using the middle cerebral artery occlusion method. The balance beam walking test was used to evaluate the symptoms of neurological deficit. MRI-T2 mapping was used to measure the damage to brain tissue. LFB staining was used to observe the damage to nerve fibers. HE staining was used to observe the damage to nerve cell, and Iba-1 and CD16/Iba-1 immunofluorescence staining were used to observe the condition of microglial activation.Results:Compared with the model group, the scores of balance beam walking ability of rats in Tanhuo Prescription high-dose group and ginaton group at 24 h, 48 h and 72 h after ischemia were significantly improved ( P<0.05, P<0.01). The scores of balance beam walking ability of rats in Tanhuo Prescription low- and medium- dose groups at 72 h after ischemia were improved ( P<0.01). Compared with the model group, the T2 values of the cortex and striatum around the infarct of rats in Tanhuo Prescription high-dose group and ginaton group were significantly reduced ( P<0.05, P<0.01), and the T2 values of the striatum around the infarct of rats in Tanhuo Prescription low- and medium- dose groups were significantly reduced ( P<0.05). Compared with the model group, the LFB IOD of the cortex, striatum and outer capsule around the infarct decreased in the Tanhuo Prescription high-,low-dose group and ginaton group ( P<0.01). The LFB IOD of striatum around infarct decreased in medium- dose Tanhuo Prescription group ( P<0.01). Compared with the model group, the pathological injury degree of the striatum around the infarct of rats in Tanhuo Prescription low- ,medium-, and high-dose groups decreased, and the cell density decreased ( P<0.05, P<0.01). The density of the cortical and striatum cells around the infarct of rats in ginaton group increased ( P<0.01, P<0.05). Compared with the model group, the number of Iba-1 and CD16/Iba-1 positive cells in the cortex and striatum around the infarct decreased in Tanhuo Prescription medium-, high-dose and ginaton groups ( P<0.01). The number of CD16/Iba-1 positive cells in the cortex and striatum around the infarct of rats in Tanhuo Prescription low-dose group decreased ( P<0.01), and the number of Iba-1 positive cells in the striatum around the infarct of rats in Tanhuo Prescription low-dose group decreased ( P<0.01). Conclusion:Tanhuo Prescription can improve the symptoms of neurological deficits in rats with cerebral ischemia, reduce the neuropathological damage in the cerebral area around ischemic infarction, and inhibit the activation of M1 microglia.
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Objective:To establish an HPLC-DAD-ELSD method for the simultaneous determination of eight main active components in Buyang Huanwu Decoction, including hydroxysafflor yellow A, paeoniflorin, calycosin glycoside, ferulic acid, ononin, calycosin, fermononetin and astragaloside.Methods:Agilent Eclipse XDB-C18 column (250 mm×4.6 mm, 5 μm) was used with acetonitrile-0.1% formic acid as the mobile phase. The flow rate was 1.0 ml/min; the column temperature was 30 ℃; the detection wavelengths were 230 nm (paeoniflorin), 254 nm (calycosin glycoside, ononin, calycosin, fermononetin), 322 nm (ferulic acid) and 403 nm (hydroxysafflor yellow A); the drift tube temperature of the evaporative light scattering detector was 60 ℃; the carrier gas flow rate was 1.6 L/min.Results:Under these conditions, the separation of hydroxysafflor yellow A, paeoniflorin, calycosin glycoside, ferulic acid, ononin, calycosin, fermononetin and astragaloside was good, and the linear relationship was in line with the requirements ( r=0.994 0-0.999 9). The average recovery was 97.8% - 101.4% ( RSD was 1.28% - 3.70%). Conclusion:The method is simple, stable and reproducible, and can be used for the quality control of Buyang Huanwu Decoction.
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Objective:To study the effect of Tanhuo Formula (THW) on the expression of Glial Fibrillary Acidic Protein (GFAP), Caspase-3 and angiogenesis.Methods:Rats were divided into sham group, model group, low-dose THW group, medium-dose THW group, high-dose THW group and Ginaton group according to random number table method. Except the sham group, rats in other groups were subjected to the middle cerebral artery occlusion via a suture method. After 2 hours,rats in the low, medium and high dose of THW groups were gavaged with 0.92, 1.84 and 3.68 g/kg of THW dry extract powder solution respectively, and the Ginaton group were gavaged with 60 mg/kg of Ginaton, once every 24 hours for 3 days. Rats in sham operation group and model group were given equal volume of normal saline by gavage. The limb symmetry score was used to evaluate limb dysfunctions. The immunofluorescence staining of GFAP and Caspase-3 were applied to assess astrocyte activation and neuronal apoptosis, respectively. The double-labeled immunofluorescence staining of platelet-endothelial cell adhesion molecule (CD31) and chondroitinsulphate peoteoglycan (NG2) were performed to detect angiogenesis.Results:Compared with the model group, rats in the high-dose of THW group showed increased limb symmetry score ( P<0.01 or P<0.05), increased number of Caspase-3 (cortex: 765.0±122.4 vs. 1 131.0±392.9; striatum: 895.9±389.8 vs. 1 401.9±453.1) ( P<0.01 or P<0.05) and CD31 +/NG2 + (cortex: 1 355.0±257.9 vs. 825.4±308.1; striatum: 1 290.9±400.9 vs. 675.2±259.7) ( P<0.01) positive cells in the periinfarct cortex and striatum, and attenuated the integrated optical density of GFAP in the perilesional cortex (4 210.00±1 226.38 vs. 7 935.78±2 001.98) ( P<0.01). Conclusions:THW could ameliorate the limb functional disorders, inhibit astroglia activation, down-regulate the expression of Caspase-3, and enhanced angiogenesis in MCAO rats.
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Pygopus(Pygo)is a new discovered component of Wnt signaling,which is located in the downstream of its core protein β-catenin and can mediate β-catenin into nucleus and activate target gene tran-scription. Pygo plays an important role in mammalian embryonic development and tumor formation. Recent studies show that Pygo regulates stem cells,tumor cells and tumor stem cells by epigenetic mechanisms such as histone interpretations and modifications. Further insights into Pygo′s functions and mechanisms will extend our knowledge of the Pygo-related tumors.