A Study On The Mechanism Of Gut Microbiota-mediated Antidepressant Effect Of Puerarin

Depression is a common recurrent chronic mental illness,mainly manifested by clinical features such as low mood,anhedonia,and suicidal tendencies in severe cases.At present,clinical first-line antidepressants still have some shortcomings,such as slow onset,unstable efficacy and large side effects,so new antidepressants need to be developed urgently.In recent years,a series of studies have shown that the inflammatory response mechanism of the “microbiota-gut-brain axis” is closely related to the occurrence of depression,and the regulation of this pathway may be a potential target for depression treatment.Puerarin has been shown to have a good antidepressant effect.Our previous studies found that it can significantly reduce the abundance of inflammation-related bacteria and correct the dysbiosis caused by stress in mice,but the mechanism related to the intestinal flora has not been fully elucidated.Therefore,this study aimed to investigate the potential link between the antidepressant effect of puerarin and the inflammatory response of the “microbiota-gut-brain axis”.First,a 4-week chronic unpredictable mild stress(CUMS)rat depression model was established,with puerarin at two doses of 30 mg/kg and 100 mg/kg,and fluoxetine as a positive control.The behavioral changes of rats were observed by open field test(OFT),sucrose preference test(SPT)and forced swimming test(FST).The fecal microflora was analyzed by 16 S r RNA sequencing.The pro-inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor alpha(TNF-α)and anti-inflammatory interleukin-10(IL-10)in hippocampus,serum and colon were detected by enzyme-linked immunosorbent assay(ELISA).The histopathological changes of colon were observed by Hematoxylin-eosin(HE)staining.Finally,Western Blot was used to determine brain-derived neurotrophic factor(BDNF),nuclear factor-kappa B(NF-κB),inhibitor a of NF-κB(IκB-α)in the hippocampus of protein expression.The results showed that puerarin could alleviate the depressive-like behaviors induced by CUMS in rats,which could significantly increase the crossing number and rearing times in the OFT,significantly increase the sugar water preference rate in the SPT,and shorten the immobility time in FST.Importantly,puerarin ameliorated CUMS-induced dysbiosis of the rat gut microbiota,such as significantly reducing the abundance of Desulfovibrio,Turicibacter,and Verrucomicrobia.In addition,it can reduce the level of pro-inflammatory factors and increase the level of anti-inflammatory factors in depressed rats,and improve the damaged colon tissue.And also found that it could up-regulate the expression of BDNF and IκB-α and inhibit the expression of NF-κB in the hippocampus.In addition,the overall antidepressant effect of puerarin 100 mg/kg dose was better than that of 30 mg/kg dose.This part of the study preliminarily explored the effect of puerarin on intestinal flora and the improvement of inflammatory reaction in CUMS rats.Next,in order to further confirm the role of gut microbiota in puerarin treatment,administered mixed antibiotics to rats by gavage and drinking water for 4 weeks to establish antibiotic-induced gut microbial depletion(AIMD)model.The same experimental method as before was used to verify whether the antidepressant effect of puerarin was mediated by gut microbiota.Interestingly,puerarin did not effectively alleviate depression-like behaviors in rats after AIMD intervention.Another important result was that the ability of puerarin to increase beneficial bacteria and decrease harmful bacteria was attenuated after AIMD intervention.In addition,it failed to effectively inhibit the inflammatory response of hippocampus,serum and colon,and could not effectively down-regulate the TLR4/NF-κB pathway.It is suggested that the intestinal flora plays an important role in the antidepressant mechanism of puerarin through the“microbiota-gut-brain axis” inflammatory response.In summary,the antidepressant mechanism of puerarin is related to the regulation of intestinal flora imbalance,the inhibition of inflammatory responses in hippocampus,serum and colon,and the downregulation of TLR4/NF-κB pathway.It can exert its antidepressant effect by effectively improving the inflammatory response of the “microbiota-gut-brain axis” pathway.This study provides certain methods and theoretical references for the study of the relationship between drugs and intestinal flora,and also provides a basis for further antidepressant research.