| Objective:The expression of CC chemokine ligand 2（CCL2）in the cerebrospinal fluid of HIV-1 infected people is significantly higher than that of normal people,and it plays an important role in the development of AIDS cognitive impairment.This experiment uses brain stereotactic technology to inject CCL2 into bilateral hippocampal regions of the rat brain to establish a rat model of cognitive impairment and use TanshinoneⅡA（Tan ⅡA）for preventive treatment.To investigate the effects of Tan ⅡA on learning memory abilities and cognitive function in rats through water maze and new object recognition experiments.Nissl staining,and TUNEL staining were used to investigate the effects of Tan ⅡA on neuronal damage and apoptosis in rats.Oxidative stress kit to investigate the effects of Tan ⅡA on the levels of superoxide dismutase（SOD）,malondialdehyde（MDA）and reduced glutathione（GSH-Px）in hippocampus of rats.Real-time PCR was used to investigate the effects of Tan ⅡA on IL-1β,IL-6,Caspase-3,Caspase-8,and Caspase-9 genes.In order to explore protective effect and its mechanism of Tan ⅡA on CCL2-induced cognitive impairment in rats and provide a certain the methods.Methods:1.70 SPF male SD rats were randomly divided into 7 groups:blank control group（control）,sham operation group（sham）,5ng CCL2 group（5ng CCL2）,memantine group（5ng CCL2+10mg/kg/d）Memantne),Tan ⅡA low dose group（5ng CCL2+25mg/kg/d Tan ⅡA）,Tan ⅡA medium dose group（5ng CCL2+50mg/kg/d Tan ⅡA）,Tan ⅡA high dose group（5ng CCL2+75mg/kg/d Tan ⅡA）,10 per group.Three days after the preventive administration of the corresponding drugs,except for the blank control group,the other groups were subjected to stereotactic brain surgery.The injection volume of each hippocampus was 2.5μL,and the injection speed was 0.3μL·min-1.The sham operation group was given the same amount of sterile saline.2.The Morris water maze experiment was performed 3-8 days after after surgery to test its learning and memory level,and then novel object recognitive test was performed to evaluate the cognitive level on 9th and 10th days.3.After the novel object recognitive test,then narcosised by 10%chloral hydrate intraperitoneal and cardiac perfusion by 4%polyformaldehyde and saline to stiffness of the limbs.The brain was quickly taken from the head and fixed by polyformaldehyde.The neuron damage and apoptosis in the hippocampal CA1region of rats were observed by Nissl staining and TUNEL staining,respectively.The remaining rats were dissected and their brains were taken out to isolate hippocampal tissue for further biochemical index detection.4.The oxidative stress kit detects the activity of oxidative stress indicators SOD,GSH-Px and the content of MDA in rat hippocampus.5.q RT-PCR was performed to detect the m RNA expression of following indexes:（1）Inflammatory cytokines IL-1β,IL-6;（2）Apoptotic-associated protein caspase-8,caspase-3,caspase-9.Results:1.Morris water maze test results:The results of the positioning sailing test showed that there was no significant difference in the average swimming speed of the rats in each group（P>0.05）.Compared with the sham operation group,the escape latency and total swimming distance of the model group were significantly longer（P<0.001）;Compared with the model group,the escape latency and total swimming distance of rats in each dose group of Tan ⅡA were significantly reduced（P<0.001）.The results of the space exploration test showed that compared with the sham operation group,the number of crossing the platform in the model group was significantly reduced（P<0.05）.Compared with the model group,the number of crossing the platform in the Tan ⅡA treatment groups was significantly increased（P<0.05）.2.New object recognition experiment results:Compared with the sham operation group,the discrimination index of the model group rats decreased significantly（P<0.05）.Compared with the model group,the discrimination index of rats in each dose group of Tan ⅡA increased significantly（P<0.05）.3.Nissl staining results:The neurons in the hippocampal CA1 region of the model group were loosely arranged,the cell morphology changed,the soma was contracted,the nucleus was deeply stained,the boundaries were unclear,and a large number of ghost cells appeared.Most of the cells in each dose group of Tan ⅡA were arranged relatively tightly,the nuclei were clear,the cell structure was intact,and the number of cell necrosis was less.4.Oxidative stress test results:Compared with the sham operation group,the activity of SOD and GSH-Px in the hippocampus of the model group was significantly reduced,and the MDA level was significantly increased（P<0.001）.In comparison,each dose group of Tan ⅡA can significantly increase the activity of SOD and GSH-Px in the hippocampus of rats,and reduce the level of MDA in the hippocampus（P<0.05,P<0.01 or P<0.001）.5.Inflammation-related gene expression results:Compared with the sham operation group,the relative expressions of IL-1βand IL-6 m RNA in the hippocampus of the model group were significantly increased（P<0.01,P<0.001）.Compared with the model group,the relative expressions of IL-1βand IL-6 m RNA in hippocampus of rats in each dose group of Tan ⅡA were significantly reduced（P<0.05,P<0.01 or P<0.001）.6.Apoptosis results of rat hippocampal tissue:（1）TUNEL staining results showed that compared with the model group,the number of tunel-positive neurons in the hippocampal CA1 area of the Tan ⅡA dose groups was significantly reduced;（2）)Apoptosis-related gene expression results show that compared with the sham operation group,the relative expression of caspase-3,caspase-8,and caspase-9 m RNA in the hippocampus of the model group was significantly increased,of which caspase-3,caspase-8 Relative m RNA expression was statistically significant（P<0.05,P<0.01）.Compared with the model group,the relative expression of caspase-3 m RNA in the hippocampal tissue of rats in each of the Tan ⅡA administration groups was significantly reduced（P<0.01）;the relative expression of caspase-8 m RNA has a decreasing trend,and the effect of Tan ⅡA in each dose group is significant,and the results are statistically significant（P<0.05）;although Tan ⅡA dose groups could reduce the relative expression of caspase-9 m RNA,there was no significant difference.Conclusion:1.Tan ⅡA can improve CCL2-induced learning memory and cognitive dysfunction.2.Tan ⅡA can participate in oxidative stress response to improve hippocampal nerve cell damage caused by CCL2 in rats.3.Tan ⅡA can regulate the inflammatory response by reducing IL-1βand IL-6inflammatory factors in the hippocampus.4.Tan ⅡA can reduce apoptosis and protect hippocampal nerve cells by regulating apoptosis factors Caspase-3,Caspase-8,and Caspase-9. |
