Study On The Antidepressant Mechanism Of Kai Xin San And Brain-targeted Delivery Of Its Active Ingredient Ginsenoside Rg1

Depression is a chronic mental disorder characterized by the lesion of brain,which threaten the health and life of human due to its high prevalence,suicide,death,and recurrence rate.The pathogenesis of depression is closely related to autophagy and inflammation.Promoting autophagy can inhibit inflammatory response,and then prevent the generating and development of depression.Kai Xin San(KXS),a traditional Chinese medicine formula firstly recorded in<Beiji Qianjin Yaofang> by Sun Simiao,is composed of four herbs: Ginseng Radix et Rhizoma,Poria,Polygala Radix,and Acori Tatarinowii Rhizoma.KXS exhibits antidepressant and anti-inflammatory effects in clinical and preclinical studies,but whether KXS exerts antidepressant effect by promoting autophagy remains unknown.The effects of ingredients of KXS on autophagy are also not clear.Therefore,it is important to clarify whether KXS can promote autophagy,and which ingredients in KXS can active autophagy.It needs a long time for KXS to generate antidepressant effect via oral or intragastric administration,which may be attributed to the poor permeability across blood brain barrier(BBB)of ingredients of KXS.Brain-targeted delivery of proautophagic active ingredients of KXS may generate antidepressant effect faster.Nano-drug delivery is an effective strategy to promote drug penetration across BBB.Borneol(BO)can open BBB,and BO modified nanocarriers exhibit good BBB penetration effect.Graphene oxide(GO)has advantages of excellent drug loading performance,easily functionalized property and good biocompatibility as nanocarrier.Moreover,GO exhibits neuroprotective effects.Therefore,it is important to construct drug delivery system based on BO and GO to deliver proautophagic active ingredients of KXS into brain for the treatment of depression.This study explored the antidepressant mechanism of KXS.Clarified the main proautophagic active ingredients of KXS based on the proautophagic antidepressant mechanism of KXS,delivered them into brain through nanocarrier,and investigated the antidepressant effect.The study contains three parts:(1)Study on the proautophagic antidepressant mechanism of KXS.It was hypothesized that KXS could inhibit NLRP3 inflammasome activation by promoting autophagy,and then produced anti-inflammatory and antidepressant effects.This hypothesis was verified by in vivo and in vitro experiments,aiming to clarify the antidepressant mechanism of KXS.Firstly,chronic unpredictable mild stress(CUMS)depression model was established,and the antidepressant effect of KXS was investigated by behavioral test.The results showed that KXS significantly improved the depressive behavior of CUMS rats.Subsequently,the effects of KXS on autophagy,NLRP3 inflammasome,inflammatory cytokine and oxidative stress were investigated in vivo and in vitro.Results in vivo demonstrated that KXS promoted the autophagy in the prefrontal cortex of rats,and inhibited the activation of NLRP3 inflammasome,inflammatory response and oxidative stress.Results in vitro showed that KXS promoted autophagy in rat astrocytes,and inhibited the activation of NLRP3 inflammasome,inflammatory response and accumulation of reactive oxygen species(ROS).The results in vitro were consistent with the results in vivo.Finally,the autophagy inhibitor 3-methyladenine(3-MA)was employed to explore whether KXS inhibited NLRP3 inflammasome activation by promoting autophagy.The results demonstrated that 3-MA diminished the inhibitory effect of KXS on NLRP3 inflammasome activation,indicating that the inhibitory effect of KXS on NLRP3 inflammasome activation was regulated by autophagy.KXS exerted anti-inflammatory and antidepressant effects by promoting autophagy.(2)Study on the proautophagic ingredients of KXS.The main proautophagic active ingredients of KXS were clarified based on the proautophagic antidepressant mechanism of KXS.Firstly,the effects of main five active ingredients of KXS(ginsenoside Rg1,ginsenoside Rb1,pachymic acid,3,6'-disinapoyl sucrose,and ?-asarone)on autophagy were investigated.The results indicated that ginsenoside Rg1 promoted autophagy.The other four ingredients did not regulate autophagy obviously.Consequently,it is preliminarily considered that ginsenoside Rg1 was the main proautophagic active ingredient of KXS.To support this conclusion,the target gene of ginsenoside Rg1 was predicted by bioinformatics analysis.Function and signaling pathway enrichment analysis of the target gene were conducted.The analysis results showed that the target gene of ginsenoside Rg1 were relevant to the biological process of autophagy,autophagosome composition,ubiquitination function and regulation of autophagy pathway.To further confirm the proautophagic activity and explore the anti-inflammatory mechanism of ginsenoside Rg1,effects of low,medium and high concentration of ginsenoside Rg1 on autophagy,NLRP3 inflammasome and inflammatory cytokine were investigated.3-MA was used to explore whether ginsenoside Rg1 exerted anti-inflammatory effect by promoting autophagy.The results demonstrated that ginsenoside Rg1 inhibited NLRP3 inflammasome activation by promoting autophagy,thus generated anti-inflammatory effect,which was consistent with the mechanism of proautophagic antidepressant effect of KXS,indicated that ginsenoside Rg1 played an important role when KXS exerted its antidepressant effect.(3)Preparation of ginsenoside Rg1 brain targeting delivery system and antidepressant study.Rg1/GO-PEG-BO nano-drug delivery system was prepared based on BO and GO,aiming to deliver ginsenoside Rg1 into brain,and exert antidepressant effect faster.GO-PEG-BO nanoccarrier was prepared by the modification of BO onto the PEG functionalized GO surface.GO-PEG-BO was characterized by infrared spectroscopy,X-ray diffraction spectroscopy,Raman spectroscopy and transmission electron microscopy.The results demonstrated that GO-PEG-BO was successfully synthesized.In vitro biocompatibility was evaluated by cytotoxicity and hemolysis assay,and in vivo biocompatibility was evaluated by histopathological analysis.The results showed that GO-PEG-BO had no obvious toxicity to h CMEC/D3 and astrocyte.The hemolysis rate was lower than 5%,indicating good in vitro biocompatibility of GO-PEG-BO.After 14 days of administration,there were no damage to the heart,liver,spleen,lung,kidney and brain of rats,demonstrating good in vivo biocompatibility of GO-PEG-BO.The BBB permeability,the mechanism of BBB crossing,and anti-inflammatory activity was evaluated in in vitro BBB model.The results showed that GO-PEG-BO had good BBB permeability and good anti-inflammatory activity.GO-PEG-BO opened BBB through opening tight junctions and inhibiting efflux pump.Cellular uptake and targeted distribution of GO-PEG-BO were investigated by fluorescence microscope and in vivo fluorescence imaging.The results indicated that GO-PEG-BO was easily internalized by astrocyte due to its inhibitory effect on efflux pump.GO-PEG-BO mainly distributed in liver and spleen.After the modification of BO,GO-PEG-BO distribution in brain was significantly enhanced,indicating good BBB permeability of GO-PEG-BO.Particle size,Zeta potential,dispersibility,loading capacity,entrapment efficiency,and in vitro release of Rg1/GO-PEG-BO were determined.The results showed that Rg1/GO-PEG-BO exhibited good dispersibility with a particle size of179.21 ± 1.95 nm,and a Zeta potential of 28.29 ± 0.43 m V.The loading capacity and entrapment efficiency of Rg1/GO-PEG-BO was 11.62 ± 0.52% and 87.58 ± 2.32%,respectively.Ginsenoside Rg1 could release from Rg1/GO-PEG-BO steadily,the cumulative release was above 90% at 36 h.The antidepressant effects were evaluated by behavioral test,and the antidepressant mechanism was verified.The results demonstrated that Rg1/GO-PEG-BO improved depressive-like behaviors thoroughly induced by CUMS after 7-day drug administration.KXS and ginsenoside Rg1 merely improved depressive-like behaviors partly.KXS and ginsenoside Rg1 generated antidepressant effect after 14-day drug administration.Rg1/GO-PEG-BO generated antidepressant effect faster than KXS and ginsenoside Rg1,and shortened treatment cycle.The antidepressant mechanism of ginsenoside Rg1 did not change after loading with GO-PEG-BO,still promoted autophagy and inhibited NLRP3 inflammasome activation.In conclusion,KXS inhibited NLRP3 inflammasome activation by promoting autophagy,and then exerted anti-inflammatory and antidepressant effects.Ginsenoside Rg1 was the main proautophagic active ingredient of KXS,and inhibited NLRP3 inflammasome activation by promoting autophagy,thus exerted anti-inflammatory effect.GO-PEG-BO had excellent BBB permeability,and Rg1/GO-PEG-BO generated antidepressant effect faster than KXS and ginsenoside Rg1.This study provides a new theoretical basis and scientific basis for the study of the antidepressant mechanism and active ingredients of KXS,and provides a new strategy for the treatment of depression.