To Investigate The Protective Effect Of Panax Notoginseng Saponins On Learning And Cognitive Impairment Induced By CCL2 In Rats And Its Mechanism Based On NMDA Receptors

Objective: To study the protective effect of Panax notoginseng saponins(PNS)on CCL2-induced cognitive impairment in rats and its molecular mechanism,and to provide new insights for the treatment of CCL2-induced learning and memory impairment.Methods:(1)80 SD male rats(200±20g)were randomly assigned to the following groups: control,sham,CCL2,CCL2+memantine(CCL2+Mem),CCL2+ifenprodil(CCL2+Ife),CCL2 + PNS in low concentration(CCL2+P-L),CCL2 + PNS in middle concentration(CCL2+P-M),and CCL2 + PNS in high concentration(CCL2+P-H)(n=10/group).The menantine(10 mg/kg/d),ifenprodil(10 mg/kg/d),PNS in low concentration(50 mg/kg/d),PNS in middle concentration(100 mg/kg/d),and PNS in high concentration(200 mg/kg/d)groups required 3 days of prophylactic administration prior to surgery.Excluding the control group,all other groups received stereotypic injection.Further,excluding the sham group,which was given ultrapure water,all other groups were subjected to bilateral hippocampal injection of 5ng of 1ng/u L CCL2.After surgery,the Morris water maze(MWM)was performed on days 6to 11,and the novel object recognition test(NORT)was performed on days 12 and 13.After NORT,all rats were sacrifificed to use the hippocampus for subsequent morphological and biochemical measurements.Nissl staining was used to observe the damage of hippocampal neurons in each group.The apoptosis of hippocampal neurons was observed by TUNEL staining.The expressions of excitatory toxicity indexes including GLAST,GLT-1 and PAG in hippocampal neurons were detected by RT-PCR.The oxidative stress stress index including SOD activity and MDA content was detected by the Elisa kit.The expression of inflammatory cytokines(IL-1β,IL-6,IL-10,IL-18)and apoptotic cytokines(Bax,Bcl-2,Caspase-3,Caspase-8,Caspase-9)in hippocampal neurons were detected by RT-PCR.(2)The effect of PNS on the excitatory postsynaptic current mediated by NMDAR(mainly NR2 AR and NR2BR)was detected by single-cell patch clamp method.Results:(1)The results of water maze showed that there was no significant difference in swimming speed among the groups(P>0.05),indicating that the stereotaxic surgery and intraperitoneal injection had no effect on the exercise ability of rats.Compared to the sham group,the escape latency and the swimming distance of CCL2 group were significantly increased(P<0.01).Compared to the CCL2 group,the escape latency and the swimming distance were significantly reduced in each administration group,and the effect of high dose PNS was similar to that of memantine.At the same time,CCL2 significantly reduced the crossing times and the time spend in the target quadrant of rats(P<0.05),while each administration group significantly increased the reduced of the crossing times and the time spend in the target quadrant of rats caused by CCL2.(2)The new object recognition ability test results showed that the discrimination index of rats in CCL2 group was significantly lower than that in the sham group(P<0.01);compared to CCL2 group,the discrimination index of the high-dose PNS group was significantly increased(P<0.01).(3)The results of Nissl staining showed that the neurons in hippocampal CA1 region of control group,sham group,CCL2+Mem group,CCL2+Ife group,CCL2+P-M group and CCL2+P-H group were well-formed and closely arranged.Compared to the sham group,the hippocampal nerve cells in CCL2 group were damaged and necrotic,and the number of healthy cells was significantly reduced(P<0.01).TUNEL staining results showed that the number of apoptotic cells in the CCL2 group was significantly increased when compared with the sham group.PNS treatment group effectively reduced cell apoptosis in a dose-dependent manner.(4)The results of kits showed that compared with the sham group,the content of MDA in the hippocampus of rats in CCL2 group was significantly increased(P<0.001),and the activity of SOD was significantly decreased(P<0.001).Compared to CCL2 group,PNS decreased the MDA content and increased the SOD activity in the rat hippocampus.(5)The results of RT-PCR showed that compared with the CCL2 group,PNS significantly increased the expression of GLT-1 and GLAST,decreased the expression of inflammatory factors(IL-1β,IL-6,IL-18,CXCL-10),and decreased the Bax/Bcl-2 ratio and the expression of apoptotic factors Caspase-3,Caspase-8and Caspase-9.(6)The results of electrophysiological experiments showed that PNS reduced the NMDAR-mediated excitatory postsynaptic current that induced by CCL2,and this effects were dose-dependent.In addition,PNS has a weak selectivity for NR2 AR or NR2B-mediated excitatory postsynaptic current.Conclusion: PNS improve CCL2-induced learning and cognitive impairment by improving glutamate metabolism and inhibiting NMDA receptor excitability,reducing neuronal excitatory toxicity,reducing intracellular oxidative stress,reducing cellular inflammation and apoptosis.