| 【Background】Schizophrenia is a serious chronic mental illness.Clinical manifestations often involve obstacles such as perception,thinking and emotion,as well as uncoordinated mental activities.Schizophrenia affects 1%of the world’s population and the lifetime prevalence of patients is0.30%～0.66%.The characteristics of high recurrence rate,high mortality and low cure rate make it be a research hotspot.Chlorpromazine（CPZ）has complex pharmacological effects,including antagonism of D2 receptors,inhibition of hypothalamic thermoregulatory centers,inhibition of serotonin receptors,antagonistic effects of adrenaline alpha receptors and M choline receptors.As the first antipsychotic drug,chlorpromazine had been widely used around the world.In addition to being used for the treatment of positive schizophrenia,CPZ can also be used clinically for sedation,antiemetics,lowering body temperature,antitumor,antituberculosis and antimicrobial,etc.However,long-term and/or excessive use of chlorpromazine can cause a variety of adverse reactions,and the number of toxic events is increasing,which has caused widespread concern in the society.Among the many adverse reactions caused by CPZ,cardiovascular reactions are the most common and the most serious,especially can cause arrhythmia and sudden cardiac death.Previous studies have shown that myotoxicity of CPZ may be related to Na+channels.Other studies have shown that the changes in the expression or function of some proteins may affect myocardial function,but these studies have not fully elucidated the myocardial toxicity and mechanism of CPZ.【Objectives】CPZ acute poisoning model was established with SD rats to calculate the LD50 of CPZ acute poisoning in rats;i TRAQ was used to comprehensively screen differentially expressed proteins in myocardium,and to explore mechanism of myocardial damage caused by CPZ poisoning based on the results combined with relevant domestic and foreign literature;In addition,Myl2 and Tpm1 in the adrenergic receptor pathway were screened according to i TRAQ results,and protein expression levels were verified by Western Blot.（1）A single intraperitoneal injection of CPZ solution was used to establish an acute poisoning model,and the behavior and histopathological changes of SD rats after the poisoning were analyzed;（2）Calculate the LD50 of CPZ acute poisoning in rats by using the point-slope method（Sun’s modified Kou’s method）combined with the method of Bliss;（3）Screen differentially expressed proteins by using i TRAQ proteomics technology;（4）To explore possible mechanisms of myocardial toxicity which caused by CPZ acute poisoning;（5）Western Blot was used to verify the expression levels of differentially expressed proteins.【Methods】（1）Establishment of experimental model:80 male and female SD rats were randomly divided into 8 groups of 10 rats each,including 7 CPZ-experimental groups（40mg/kg,56mg/kg,80mg/kg,113 mg/kg,159 mg/kg,225 mg/kg,318 mg/kg）and a blank control group（normal saline）.A single intraperitoneal injection was used to rats in each experimental group with CPZ saline solution（1ml per 100g）at the corresponding concentration,and the behavioral changes of rats after exposure were observed;the blank group was given a single saline solution（1 m L/100g）intraperitoneally.The rats that died within one week were immediately dissected,the heart was quickly extracted,the saline was rinsed and weighed,the apical tissue was cut,frozen in liquid nitrogen,and then stored at-80°C.The remaining myocardial tissue was fixed with paraformaldehyde and made into sections for examination.After the rats were observed and survived for one week,they were sacrificed by cervical dislocation,and the dissection was performed as before.（2）Determination of LD50:According to the data obtained by the exposure group,the point slant method（Sun’s modified Kou’s method）and Bliss method were used to calculate the LD50 of CPZ acute poisoning;（3）Nine SD rats were randomly divided into 3 groups,including the LD5 dose experimental group,the LD50 dose experimental group and the blank control group.The rats were sacrificed after 24 hours of breeding.The apical tissue of each group of rats was extracted and myocardial proteins were extracted and applied.i TRAQ proteomics method was used to detect differentially expressed proteins in the heart muscle;（4）Classify the selected target proteins,we explored the myocardial toxicity of CPZ rats combined with the relevant literature,and then analyze the possible myocardial toxicity of CPZ.（5）Western Blot was used to verify the protein expression levels of Myl2 and Tpm1 in the adrenergic receptor pathway.【Results】（1）Observation of poisoning symptoms:The rats in each CPZ exposure experimental group showed the following symptoms,including drowsiness,slow response,muscle relaxation,abdominal breathing,and decreased body temperature;The male rats showed testicular swelling.Some rats also exhibited symptoms such as claws showing internal rotation,incontinence,tearing,bleeding around the eyes,bleeding from the tips of the claws,etc;（2）Histological and pathological changes:There were no obvious pathological changes in the myocardium of the rats with naked eyes,punctate sarcoplasmic agglutination was observed on histological examination,and focal myocardial hemorrhage and the infiltration of inflammatory cells were seen in some rats.Partial rat anatomy showed pulmonary hemorrhage and urine retention;（3）Calculating the LD50:The point oblique method（Sun’s modified Kou’s method）combined with Bliss’s method were used to calculate the LD50 of rat that handled by acute intraperitoneal injection.（4）Screening of differentially expressed proteins:i TRAQ technology combined with liquid chromatography-tandem mass spectrometry（LC-MS/MS）was used to detect differentially expressed proteins in myocardium of CPZ acutely poisoned rats.There were4463 kinds of them,among which there were 870kinds of statistically significant proteins（p<0.05）.（5）Compared with the control group,the expression level of Myl2 increased significantly in all concentration of CPZ（P<0.05）,which consistent with the trend of i TRAQ results;the expression level of Tpm1 in the exposed group was higher than that of the control group,but the expression level decreased with the increase of CPZ exposure concentration,indicating that the expression level of Tpm1 increased and then decreased with the increase of CPZ concentration,which was consistent with the trend of i TRAQ results.【Conclusion】（1）Equal volume（1m L/100g）intraperitoneal injection of CPZ saline solution was used to establish a model of CPZ acute poisoning in rats successfully.After acute exposure to CPZ in rats,drowsiness,muscle relaxation,and decrease in body temperature rapidly occurred.Male rats developed testicular swelling;（2）Calculate the LD50 of 94 mg/kg based on the mortality of a single intraperitoneal injection of CPZ saline solution in rats;（3）By using i TRAQ combined with LC-MS/MS proteomics technology,we screened870 proteins with statistically significant myocardial differential expression;（4）Research and analysis of differentially expressed proteins in myocardium of CPZ acutely poisoned rats,and we found significant differences in cardiac adrenergic signaling pathway,calcium signaling pathway,arrhythmogenic right ventricular cardiomyopathy（ARVC）and myocardial contraction related proteins Sexual expression,these proteins may be markers of myocardial toxicity of CPZ;（5）The protein expression levels of Myl2 and Tpm1 in the adrenergic receptor pathway were verified by Western Blot.Myl2 expression increased with the increase of CPZ concentration,and Tpm1 increased with the CPZ concentration.The expression increased first and then decreased.The expression of the two differential proteins was consistent with the i TRAQ result trend,indicating that the i TRAQ result was reliable. |
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