| Objectives:To explore the intervention effect of cannabidiol(CBD)on neuronal apoptosis-related proteins(Caspase-3,Cleaved caspase-3,Bax,Bcl-2)after methamphetamine(METH)addiction.To further explore whether the molecular mechanism of neuronal apoptosis induced by CBD addiction to METH involves the Keap1-Nrf2 signaling pathway.Methods:The METH addiction model of C57BL/6 mice was established by alternate intraperitoneal injection of METH and normal saline.Experimental mice were randomly divided into five groups: normal group(Control group),addiction model group(METH+NS group),CBD low-dose intervention group(METH+CBD 5mg/kg group),CBD middle-dose intervention group(METH+CBD 10mg/kg group),CBD high-dose intervention group(METH+CBD 20mg/kg group).Using conditioned place preference(CPP)score and Western Blot experiment of apoptotic protein in four brain regions of prefrontal cortex(PFC),nucleus accumbens(NAc),hippocampus(HPC)and ventral tegmental area(VTA),to preliminarily explore whether CBD has an intervening effect on apoptosis,and at the same time determine the optimal dose of CBD intervention.Randomly divided experimental mice into normal group(Control group),addiction model group(METH+NS group)and CBD optimal dose intervention group(METH+CBD 10mg/kg group),select four brain regions of PFC,NAc,HPC,VTA,using HE staining,Nissl staining,immunohistochemical staining,immunofluorescence double-labeled staining and RT-q PCR experiments,it is clear that CBD can reduce neuronal apoptosis caused by METH addiction.Experimental mice were randomly divided into normal group(Control group),addiction model group(METH+NS group),CBD optimal dose intervention group(METH+CBD 10mg/kg group)and Nrf2 inhibitor ML385 group(METH+CBD+ML385 group).Select the nucleus accumbens(NAc)brain region,and explore the relationship between the intervention effect of CBD on METH-induced neuronal apoptosis and the Keap1-Nrf2 signaling pathway through immunofluorescence double-labeled staining,Western Blot,RT-q PCR and ELISA experiments mechanism.Results:Part 1 Intervention of CBD on neuronal apoptosis induced by METH and determination of the optimal dose of CBD intervention1.CPP score results:(1)After METH addiction,the CPP score increased(P < 0.01).(2)The CPP score decreased slightly after CBD 5mg/kg intervention(P > 0.05),and the CPP score decreased significantly after CBD 10mg/kg and CBD20mg/kg intervention(P < 0.01),among which the CPP score of CBD 10mg/kg intervention lowest.2.Western Blot results:(1)After METH addiction,the expression of apoptosis proteins Caspase-3,Cleaved caspase-3 and Bax in PFC,NAc,HPC and VTA all increased(P < 0.05);The expression of death protein Bcl-2 decreased(P < 0.05).(2)After CBD intervention:(1)Apoptotic protein Caspase-3 decreased in PFC,NAc and HPC except VTA after CBD 5 mg/kg intervention(P < 0.05);after CBD 10 mg/kg intervention,PFC,NAc,HPC and VTA decreased(P < 0.05);after CBD 20 mg/kg intervention,there was a decrease in PFC and NAc(P < 0.05)except for HPC and VTA.Comprehensive analysis showed that the expression of Caspase-3 protein was the lowest in the four brain regions of PFC,NAc,HPC and VTA after CBD 10mg/kg intervention.(2)The apoptotic protein Cleaved caspase-3 decreased only in NAc after CBD 5 mg/kg intervention(P < 0.05);after CBD 10 mg/kg intervention,all decreased in PFC,NAc,HPC and VTA(P < 0.05);In addition to NAc and VTA,decreases were seen in PFC and HPC after CBD 20 mg/kg intervention(P < 0.05).Comprehensive analysis showed that the expression of Cleaved caspase-3protein was the lowest in the three brain regions of NAc,HPC and VTA after CBD 10mg/kg intervention.(3)The apoptotic protein Bax decreased in NAc and VTA except PFC and HPC after CBD 5 mg/kg intervention(P < 0.05);after CBD 10 mg/kg intervention,PFC,NAc,HPC and VTA decreased(P < 0.05);after CBD 20mg/kg intervention,except for NAc,there were decreases in PFC,HPC and VTA(P < 0.05).Comprehensive analysis showed that the expression of Bax protein was the lowest in PFC,NAc and VTA three brain regions after CBD 10mg/kg intervention.(4)The anti-apoptotic protein Bcl-2 increased in NAc and VTA except PFC and HPC after CBD 5 mg/kg intervention(P < 0.05);after CBD 10 mg/kg intervention,it appeared in PFC,NAc,HPC and VTA Elevated(P < 0.05);increased(P < 0.05)in NAc,HPC and VTA,except PFC,after CBD 20 mg/kg intervention.Comprehensive analysis showed that the expression of Bcl-2 protein was the highest in the three brain regions of PFC,NAc and HPC after CBD 10mg/kg intervention.The results of the above CPP score and Western Blot experiment showed that CBD has an inhibitory effect on apoptosis caused by METH addiction.At the same time,the optimal intervention dose of CBD was determined to be 10 mg/kg for follow-up experiments.3.HE staining results:(1)After METH addiction,the arrangement of neurons in PFC,NAc,HPC and VTA was disordered and there was no sense of hierarchy.Some neurons suffered from pyknotic necrosis,and the nuclei and nucleoli disappeared.Cells condense into irregular,triangular,and deeply stained forms.(2)After the intervention of CBD 10mg/kg,the pathological changes of cells were alleviated.4.Results of Nissl staining:(1)After METH addiction,the number of neurons with pyknotic necrosis in PFC,NAc,HPC and VTA increased(P < 0.01).(2)After CBD 10mg/kg intervention,the neuron counts of pyknotic necrosis in the four brain regions decreased(P < 0.01).5.Immunohistochemical staining results:(1)After METH addiction,the positive expressions of apoptotic proteins Caspase-3,Cleaved caspase-3 and Bax increased in PFC,NAc,HPC and VTA(P < 0.05),while The positive expression of anti-apoptotic protein Bcl-2 decreased(P < 0.05).(2)After CBD 10mg/kg intervention,the positive expression of Caspase-3,Cleaved caspase-3 and Bax in the four brain regions decreased(P < 0.05);the positive expression of Bcl-2increased(P < 0.05).6.Immunofluorescence double-labeled staining results:(1)After METH addiction,in PFC,NAc,HPC and VTA,apoptotic proteins Cleaved caspase-3,Bax and Neu N(neuron marker)fluorescent double-labeled co-expressed cells The counts of were increased(P < 0.01).(2)After CBD 10mg/kg intervention,the counts of Cleaved caspase-3,Bax and Neu N co-expressed cells in the four brain regions decreased(P < 0.01).7.RT-q PCR experiment results:(1)After METH addiction,the m RNA expressions of apoptosis proteins Caspase-3 and Bax increased in PFC,NAc,HPC and VTA(P< 0.05);anti-apoptosis protein Bcl-2 m RNA expression decreased(P < 0.05).(2)After CBD 10mg/kg intervention,the m RNA expression of Caspase-3 and Bax in the four brain regions decreased(P < 0.05);the m RNA expression of Bcl-2increased(P < 0.05).Part 2 Study on the mechanism of CBD intervention and Keap1-Nrf2 signaling pathway8.Immunofluorescence double-labeled staining results:(1)After METH addiction,the number of cells co-expressing the signaling pathway Nrf2 protein and Neu N(neuron marker)in the nucleus accumbens(NAc)decreased(P < 0.01),the count of cells co-expressed with Keap1 protein and Neu N fluorescence double labeling pathway increased(P < 0.01).(2)After CBD 10mg/kg intervention,the counts of Nrf2 and Neu N co-expressed cells increased(P < 0.01),and the counts of Keap1 and Neu N co-expressed cells decreased(P < 0.01).(3)After ML385 inhibition,the count of Nrf2 and Neu N co-expression cells decreased(P < 0.01),and the count of Keap1 and Neu N co-expression cells increased(P < 0.01).9.Western Blot results:(1)After METH addiction,the protein expression of signaling pathway Nrf2 in the nucleus accumbens(NAc)decreased(P < 0.01),and the expression of Keap1 protein increased(P < 0.01);The protein expression of Caspase-3,Cleaved caspase-3 and Bax increased(P < 0.01),and the expression of anti-apoptotic protein Bcl-2 decreased(P < 0.01).(2)After CBD 10mg/kg intervention,the expression of Nrf2 protein increased(P < 0.05),the expression of Keap1 protein decreased(P < 0.05);the expression of Caspase-3,Cleaved caspase-3 and Bax protein decreased(P < 0.05).0.01),the expression of Bcl-2protein increased(P < 0.01).(3)After the intervention of ML385 inhibitor,the expression of Nrf2 protein decreased(P < 0.05),the expression of Keap1 protein increased(P < 0.05);the expression of Caspase-3,Cleaved caspase-3 and Bax protein increased(P < 0.05),Bcl-2 protein expression decreased(P < 0.01).10.RT-q PCR experiment results:(1)After METH addiction,the m RNA expression of signaling pathway protein Nrf2 in the nucleus accumbens(NAc)decreased(P <0.01),and the m RNA expression of Keap1 increased(P < 0.01);The m RNA expression of apoptotic proteins Caspase-3 and Bax increased(P < 0.01),and the m RNA expression of anti-apoptotic protein Bcl-2 decreased(P < 0.05).(2)After CBD 10mg/kg intervention,the m RNA expression of Nrf2 increased(P < 0.01),the m RNA expression of Keap1 decreased(P < 0.01);the m RNA expression of Caspase-3 and Bax decreased(P < 0.01),the m RNA expression of Bcl-2increased(P < 0.01).(3)After ML385 inhibition,the m RNA expression of Nrf2decreased(P < 0.01),the m RNA expression of Keap1 increased(P < 0.01);while the m RNA expression of Caspase-3 and Bax increased(P < 0.01),the m RNA expression of Bcl-2 decreased(P < 0.01).11.The results of serum ELISA detection of reactive oxygen species(ROS),reduced glutathione(GSH),and superoxide dismutase(SOD):(1)After METH addiction,the content of oxidative stress product ROS increased significantly(P < 0.05),the content of antioxidants GSH and SOD decreased(P < 0.05).(2)After CBD10mg/kg intervention,the ROS content decreased(P < 0.05),and the GSH and SOD content increased(P < 0.05).(3)After the intervention of ML385 inhibitor,the content of ROS increased significantly(P < 0.05),and the content of GSH and SOD decreased(P < 0.05).Conclusions:1.CBD can downregulate the expression of apoptotic proteins Caspase-3,Cleaved caspase-3 and Bax in METH addicted mice,upregulate the expression of anti apoptotic protein Bcl-2,and inhibit neuronal apoptosis.2.CBD can regulate oxidative stress through the Keap1-Nrf2 signaling pathway to inhibit neuronal apoptosis and play a certain role in neuroprotection. |
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