Studies On Material Basis Of Gualou Guizhi Decoction Treating Spasticity After Stroke

Background:Stroke, the characteristics of high prevalence, high morbidity and high mortality. Muscle spasm after stroke is a common sequela, seriously affected the patient’s rehabilitation and daily life ability. At present the treatment drugs of muscle spasm after stroke was relatively scarce.The understanding of stroke has a long history of traditional Chinese medicine which shows good curative effect advantage in the treatment of chronic, multi-factor complex diseases. According to the original prescription from the "Jinkui Yaolve" (one of the Chinese traditional medical classics), the prescription of Gualou Guizhi decoction (GLGZD) comprised radix trichosanthis, radix paeoniae alba, ramulus cinnamomi, rhizoma zingiberis recens, radix glycyrrhizae, Fructus Ziziphi Jujubae. It has the efficacy of relieving muscle, removing the pathogens and relieving channels,used to treating muscle spasm. The preliminary clinical research shown that Gualou guizhi decoction (GLGZD) has good curative effect of spasm after stroke,while its efficacy material base is unclear. The study was funded by the Important Subject of Fujian Province Science and technology hall of China (Item number:2012Y0041).Objective:This research aims to interpret the efficacy material base of muscle spasm after stroke, discussion its brain targeting advantages and provide powerful scientific data to support for multiple targets of treatment in the aspect of cerebrovascular disease and the efficacy of traditional Chinese medicine compound material basic research.Methods:(1)Using the method of liquid chromatograph-mass spectrometer for determination of two kinds of amino acids(citrulline and gamma aminobutyric acid) and other 24 kinds of active ingredients in GLGZD.(2) Focal cerebral I/R injury in rats was induced by middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion. In order to explore therapeutic effects of GLGZD treating muscle spasm after stroke, the neurologic impairment, cerebral infarction volume histopathological changes and the neurotransmitter content of cerebrospinal fluid were measured,(3) Using UPLC-MS method to the analysis and detection the composition into the brain after ischemia reperfusion model rats which were given GLGZD.Using GC-MS method to the analysis and detection the volatile composition into the brain after ischemia reperfusion model rats which were given GLGZD.(4)The method of evans blue staining was used to observing the changes of BBB permeability after the treatment; the immunohistochemical technique was used to detecting the expression of the tight junction protein ZO-1、Claudin-5 and the water channel protein AQP-4; the ELISA method was measured the change of matrix metalloproteinases in brain tissue of the MMP-2 and MMP-9.Results:(1) Respectively established HPLC-MS, UPLC-MS/MS method for determination of two kinds of amino acids(citrulline and gamma aminobutyric acid) and other 24 kinds of active ingredients in GLGZD, including Gallic Acid、Protocatechuic Acid、Oxypaeoniflorin、 Catechinu、4-Hydroxybenzoic acid、Methyl Gallate、Vanillic Acid、Albiflorin、Paeoniflorin、 4-Hydroxycinnamic Acid、Liquiritin、Astragalin、3-Hydroxycinnamic Acid、Isoliquiritin、 2-Hydroxycinnamic Acid、Liquiritigenin、Jujuboside A、Cinnamic Acid、Glycyrrhizic Acid、 6-Gingero1、Licochalcone A、8-Gingerol、6-Shogaol and Glycyrrhetinic Acid.(2) The pharmacodynamic results show that in terms of neurologic symptom scores,the seventh day score of low, medium and high dose GLGZD group below MCAO model group (P<0.05);In obstacles of motor function aspect, compared with MCAO model group, the score of low, medium and high dose GLGZD group significantly increased(P<0.05). In cerebral infarction volume; MCAO model group compared with GLGZD high and medium dose groups had significant difference (P<0.05);In terms of brain tissue pathology, dosage group compared with MCAO model group, the pathological change significantly reduce. Although neuronal degeneration, necrosis, tissue edema, arrange scattered cells, karyolysis and nucleus pycnosis, but the degree and quantity are less MCAO model group.In the aspect of amino acid neurotransmitter, compared with MCAO model group,the high dose of GLGZD group can significantly reduce the content of amino acid neurotransmitter (aspartic acid, glutamic acid, glycine) in the brain tissue.(3) By UPLC-MS method, the eleven compositions in the cerebrospinal fluid and brain tissue were identified after lavage administration GLGZD, contains citruline, gallic acid, peoniflorin, liquiritin apioside, liquiritin, alibiflorin, isoliquiritin apioside, isoliquiritin,liquiritigenin, lsoliquiritigenin and glycyrrhizic acid.By GC-MS method, ninety-three volatile components were identified in GLGZD and preliminarily identified fifty-four volatile into the brain.(4) In brain tissue EB content aspect, compared with MCAO model group,the GLGZD soup brain tissue EB content were lower (P<0.01).Immunohistochemical results show that Zo-1、 Claudin-5 main position in the cytoplasm, Compared with the sham-operated group, Zo-1、 Claudin-5 protein expression were significantly decreased in model group, while AQP-4 protein expression were significantly increased (P<0.01).The expression of Zo-1、Claudin-5 protein was increased obviously in the GLGZD group, while AQP-4 protein expression were significantly decreased (P<0.01).In the aspect of MMP-2 and MMP-9 content, Compared with the sham-operated group, the content of MMP-2 and MMP-9 increased significantly in the model group (P<0.01,P<0.001); Compared with the model group, the content decreased significantly in the GLGZD group (P< 0.01, P< 0.001).Conclusion:(1) The method of HPLC-MS, UPLC-MS/MS for determination of two kinds of amino acids (citrulline and gamma aminobutyric acid) and other 24 kinds of active ingredients in GLGZD has the advantages of short analysis time and high detection sensitivity and laid a solid basis for study of efficacy material base.(2) GLGZD could improve the neurological performance and cerebral pathological changes, alleviate obstacles of motor function,reduce infarct volumes and reduce the content of amino acid neurotransmitter in the brain tissue (aspartic acid, glutamic acid, glycine) as compared with the MCAO group.(3) Contains citruline, gallic acid, peoniflorin, liquiritin apioside, liquiritin, alibiflorin, Isoliqui-ritin apioside, isoliquiritin,liquiritigenin, lsoliquiritigenin,glycyrrhizic acid and 54 kinds of volatile components have been identified, could pass through brain tissue.These composition may be pharmaceutical material basis that play a role of nerve protective of cerebral ischemia reperfusion injury in rats.(4) GLGZD Can reduce the permeability of BBB. Its mechanism may be through restore the function of tight junction, by raising the protein expression of ZO-1、Claudin-5; cut the water channel protein expression of AQP4, inhibit metal matrix protein MMP-2 and MMP-9, reduce ischemia reperfusion after brain edema and nerve cell damage degree,and then to play multiple targets for brain protection,indirectly relieved the muscle spasm after stroke.