Discussion On The Mechanism Of Guipi Decoction On Autism Based On CD38 Signal Pathway

Objective: To investigate the effect of Guipi decoction on ICR mice and CD38 KD mice and the mechanism of treating autism.Methods: The wild-type ICR mice were subjected to CD38 gene knockdown(KD)by gene silencing technique to establish a CD38 KD mice model.According to the weight mice were randomly divided into several groups:control group,empty vector group,model group,oxytocin(OT)group,high-dose Guipi Decoction group,middle-dose Guipi Decoction group and low-dose Guipi Decoction group.The control group,empty vector group and model group were given the same volume of pure water by gavage,the OT group was given OT by intraperitoneal injection at the dose of 100ng/kg,the high-dose Guipi Decoction group was given Guipi Decoction at the dose of3.4g/kg,the middle-dose Guipi Decoction group was given Guipi Decoction at the dose of 1.7g/kg,and the low-dose Guipi Decoction group was given Guipi Decoction at the dose of 0.85g/kg.The administration volume of the above groups were 10 m L/kg per mice each time.The mice in each group was given the drug twice a day for one week,fasting for 24 hours on the last day,and the experiment was carried out 2 hours after the last administration.Tail suspension test,open field test,elevated-plus maze test and three chamber test were used to detect the changes of social behavior in mice.ELISA was used to detect the expression level of OT in cerebrospinal fluid and plasma.Western blot was used to detect the expression level of CD38 protein in hypothalamus.Immunohistochemistry was used to detect the expression level of c-fos in hypothalamus.Results:1.Effect of Guipi Decoction on ICR miceThe results of open-field test showed that OT and Guipi Decoction had the tendency to improve the autonomous activity,explore new environment of ICR mice,but there was no significant difference between groups.The results of elevated plus-maze test showed that OT and Guipi Decoction had the tendency to reduce the numbers,the retention time and their percentage of ICR mice entering the closed arm,especially the low-dose Guipi Decoction could significantly reduce the percentage of retention time(P<0.05).The results of the three-chamber test showed that OT and Guipi Decoction had no significant effect on the social behavior of ICR mice.ELISA results showed that OT intervention could significantly increase the content of OT in plasma or cerebrospinal fluid of ICR mice(P<0.01 or P<0.05),the low-dose and the high-dose of Guipi Decoction intervention can significantly increase the content of OT in CSF(P<0.05),but had no significant effect on OT in plasma.Western blot and immunostaining results showed that the intervention of OT and Guipi Decoction had no significant effect on the expression of CD38 protein in the hypothalamus and the expression of c-fos in the hypothalamus of ICR mice.2.The effect of Guipi Decoction on CD38 KD miceCompared with the blank group,the expression of CD38 protein in the hypothalamus of CD38 KD model mice was significantly reduced(P<0.01).Plasma and cerebrospinal fluid OT showed a downward trend,and the OT of cerebrospinal fluid decreased significantly(P<0.01).The immobility time of the tail suspension test was significantly reduced(P<0.01).The total distance,active time,and central residence time ratio of the open-field test were significantly reduced(P<0.01).The corner residence time ratio and peripheral residence time ratio both increased significantly(P<0.01).In the elevated plus maze test,the percentage of open-arm entries and the percentage of open-arm residence time were significantly reduced(P<0.01),and the percentage of closed-arm residence time was significantly increased(P<0.01).The total distance of the three-chamber test was significantly reduced(P<0.05),and the index value and delta value of the exploration time of the stranger2 mice were significantly reduced(P<0.01).After administration intervention,compared with CD38 KD model mice.The results of the open-field test showed that after OT intervention,CD38 KD mice’s total distance was increased significantly,the activity time and central residence time were increased significantly(P<0.01 or P<0.05).After the intervention of the middle and high doses of Guipi Decoction,the total distance of CD38 KD mice was increased significantly(P<0.01).The ratio of residence time in corners and the ratio of peripheral residence time of the middle and high doses of Guipi Decoction were decreased significantly(P<0.01).The peripheral residence time ratio of the low-dose Guipi Decoction was significantly reduced(P<0.01).The results of the elevated plus-maze test showed that the percentage of open arm residence time of CD38 KD mice was significantly increased after OT administration(P<0.05).The percentage of residence time with closed arm was significantly reduced(P<0.05).Guipi Decoction low and middle dose intervention can significantly increase the percentage of open arm entries and open arm residence time percentage(P<0.05 or P<0.01)in CD38 KD mice,while the percentage of closed arm residence time decreased significantly(P<0.01).The results of the three-chamber test showed that the total distance of CD38 KD mice increased significantly after OT intervention(P<0.001).The index value and delta value of the exploration time of the stranger2 mic were significantly increased(P<0.05 or P<0.01).After the intervention of Guipi Decoction,the total distance of CD38 KD mice showed an increasing trend.Among them,the low and middle doses of Guipi Decoction can significantly increase the total distance(P<0.01).Middle and high doses can significantly increase the index value of the exploration time of stranger2 mice(P<0.05 or P<0.01).The Guipi Decoction can increase the delta value of exploring time to stranger mice(P<0.01).The ELISA results showed that the cerebrospinal fluid and plasma OT secretion of CD38 KD mice increased after the administration of OT and Guipi Decoction,and the OT content of the cerebrospinal fluid increased significantly(P<0.05 or P<0.01).The western blot results showed that the intervention of OT and Guipi Decoction can significantly improve the expression of CD38 protein in the hypothalamus of CD38 KD mice(P<0.01).The immunostaining results showed that the intervention of OT and the high-dose of Guipi Decoction can reduce the expression of c-fos in the hypothalamus of CD38 KD mice(P<0.05 or P<0.01).Conclusion:1.The levels of OT in plasma and cerebrospinal fluid of CD38 knockdown mice decreased,and they showed abnormal social behaviors of autism,such as autonomic activity disorder,exploration behavior disorder,sociability disorder and anxiety;2.Guipi Decoction can improve ICR mice autonomic activity,exploration ability,anxiety behavior and OT secretion;3.Guipi Decoction can improve CD38 KD autism-like social behavior disorder,and the level of OT in cerebrospinal fluid was significantly increased,which may be through the hypothalamus CD38 signaling pathway to promote OT secretion to improve the autistic behavior of CD38 KD model mice;4.Guipi Decoction may have other pathways involved in improving autism-like behavior,which needs further research and discussion.