| Cerebral palsy（CP）is a central nervous system disease characterized by movement disorders.The pathogenesis of cerebral palsy is very complex,and there is no clear understanding of it.The high-risk factors of cerebral palsy include intrauterine infection,premature birth,hypoxiaischemia,hyperbilirubinemia and so on.From 2009 to 2016,the American epidemiological survey found that 3.2 cases per 1000 newborns suffered from cerebral palsy.The high prevalence of cerebral palsy makes it a world public health priority for prevention.At present,the diagnosis process of cerebral palsy is complicated and takes a long time,which may delay the best time for treatment for children.At the same time,the effect of modern clinical treatment of cerebral palsy is not significant,and the side effects of drug treatment are relatively large.Therefore,it is very urgent and necessary to explore the possible pathogenesis of cerebral palsy,to find a new rapid and accurate diagnosis method for cerebral palsy,and to find drugs with good effect and less side effects.Liuwei Dihuang Pill,a classic Chinese prescription,has undergone long-term clinical application and extensive research in nourishing kidney essence,strengthening bones,and delaying aging.A few animal experimental studies have shown that Liuwei Dihuang Pill can inhibit membrane excitability and membrane transport,increase the expression of BDNF,repair the cerebral cortex,regulate monoamine neurotransmitters,improve rat motor function,reduce neuron loss,and restore the neuroendocrine system and other pharmacological functions.However,there are few studies on the improvement of cerebral palsy by Liuwei Dihuang Pill,and there is no systematic interpretation of the mechanism of Liuwei Dihuang Pill in improving cerebral palsy.In this study,metabolomics technology was applied to the study of cerebral palsy,and multivariate data analysis was performed on cerebral palsy rat plasma based on UPLC-Q-TOFMS to find cerebral palsy-related biomarkers and reveal the possible pathogenesis of cerebral palsy.And explore the improvement mechanism of Liuwei Dihuang Pill on cerebral palsy.The main content of this article includes the following three parts:（1）Establishment of cerebral palsy model rats induced by lipopolysaccharide combined with hypoxia;（2）Study on the improvement effect of Liuwei Dihuang Pill on cerebral palsy rats;（3）Plasma metabolomic analysis on the improving effect of Liuwei Dihuang Pills on cerebral palsy rats.First,the model was established by combining prenatal injection of lipopolysaccharide in pregnant rats,perinatal anoxia and sensorimotor restriction in neonatal rats.The three aspects of body weight,motor skill and cerebral cortex histopathology were used as the evaluation criteria for the success of the modeling,and the improvement effect of Liuwei Dihuang Pill on cerebral palsy model rats was explored.The results of body weight analysis showed that the rats in the control group were heavier than the model group,while the body weight of the Liuwei Dihuang Pill group could return to normal levels after administration.The results of motor skill test showed that compared with the control group,the rats in the model group had more errors in the horizontal ladder test and abnormal behavior and posture in the narrow suspended bar test.After administration,the number of errors in the Liuwei Dihuang Pill group decreased,and behavior and posture returned to more normal levels.In histopathology,compared with the control group,the Nissl bodies in the model group were dissolved,the number of Nissl bodies was reduced,and the neuronal cells were destroyed.After administration,the shape and number of nissl bodies in the Liuwei Dihuang Pill group returned to normal,and the neurons were relatively intact.These three experiments demonstrated that lipopolysaccharide combined with hypoxia-induced cerebral palsy model was successfully modeled,and Liuwei Dihuang Pills had improved effects on body weight,behavior and posture,nissl bodies,and neurons.Subsequently,the plasma metabolic profiles of rats in each group were studied based on UPLC-Q-TOF-MS.The results showed that there were significant differences in metabolic patterns among the three groups,and the Liuwei Dihuang Pill group tended to return to the control group.43 endogenous metabolites were identified as potential biomarkers related to cerebral palsy,28 metabolites were detected in positive ion mode and 15 metabolites were detected in negative ion mode.Compared with the control group,the levels of Oleamide,11,14,17-Eicosatrienoic acid,13-oxoode,Oleic acid,PS（18:0/20:4（8Z,11 Z,14Z,17Z））,PC（15:0/18:2（9Z,12Z））,L-Malic acid,Stearic acid,Oxypurinol,L-Acetylcarnitine,Uric acid,Niacinamide,Sphingosine 1-phosphate,Pregnenolone,2-Hydroxy-6-pentadecylbenzoic acid,Palmitic acid,D-Galactose,Lyso PC（16:0）,Glyceric acid,4-Oxoretinol,13S-hydroxyoctadecadienoic acid,Prostaglandin F1 a,3-ketoDigoxigenin,9,10,13-Tri HOME,17-Hydroxylinolenic acid,1D-Myo-inositol 1,3-bisphosphate,Oxidized glutathione,Adenosine monophosphate,Cortolone,Heme,（9S,10S）-10-hydroxy-9-（phosphonooxy）octadecenoate,Tetrahydrodeoxycortisol,Xanthosine and Prostaglandin E2 in the model group decreased,while the levels of Deoxycholic acid glycine conjugate,2-Phenylethanol glucuronide,Glycocholic acid,Cervonoyl ethanolamide,Adrenic acid,Palmitaldehyde,Vitamin A,Taurodeoxycholic acid and Hippuric acid increased.After administration of Liuwei Dihuang Pills,the contents of these metabolites tended to return to normal levels.Meanwhile,cluster analysis classified the control group and the Liuwei Dihuang Pill group into one category,indicating that the metabolic characteristics of the two groups were similar.Metabolic pathway analysis showed that these biomarkers mainly affected Glycerophospholipid metabolism,Glyoxylate and dicarboxylate metabolism,Purine metabolism,Nicotinate and nicotinamide metabolism,Glycerolipid metabolism,Pentose and glucuronate interconversions,Citrate cycle（TCA cycle）,Sphingolipid metabolism,Pyruvate metabolism,Galactose metabolism,Phosphatidylinositol signaling system,Glutathione metabolism,Porphyrin and chlorophyll metabolism,Glycine,serine and threonine metabolism.Finally,combined with the biological significance of these differential metabolites,we speculate that cerebral palsy may be caused by inflammation in neonatal children leading to brain damage,changes in membrane fluidity and permeability,abnormal energy metabolism,cellular oxidative damage caused by free radicals,and apoptosis of neuronal cells.In addition,Liuwei Dihuang Pills can improve cerebral palsy by inhibit inflammation in the body,activate the peripheral cholinergic neuron system and regulate the central nervous system,regulate energy metabolism,scavenge free radicals,protect cells from damage,protect nerve cells and regulate sphingolipid metabolism pathways,promoting the formation of myelin.This experiment identified potential biomarkers related to cerebral palsy based on metabolomics and explored the possible pathogenesis of cerebral palsy and the improvement mechanism of Liuwei Dihuang Pill on cerebral palsy.We hope this work can provide some references for the early diagnosis and treatment of cerebral palsy in the future.In addition,the study on the improvement mechanism of Liuwei Dihuang Pill can provide a theoretical basis for its application in cerebral palsy,and its specific mechanism of action needs further research. |
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