| Kai-xin-san (KXS) is a famous Chinese medicinal formula firstly prescribed by Sun Simiao in Beiji-qianjin-yaofang in 1400 years ago. It consists of four herbs: Ginseng Radix et Rhizoma, Polygalae Radix, Acori Tatarinowii Rhizoma and Poria. Interestingly, the ratio is distinct in different ancient medical books, and three of them are widely used today: (i) K-652, named after the recorded year in 652 A.D., with a ratio of 1:1:25:50; (ii) K-984, recorded in 984 A.D., with a ratio of 1:1:1:2; and (iii) D-652, recorded in 652 A.D., with a ratio of 3:2:2:3. All of them are used to treat stress-related psychiatric disease with the symptoms such as depression, forgetfulness and dizziness. However, the exact biological effects and action mechanisms of these herbal mixtures are not known yet. Here, we aimed to reveal the action mechanism of KXS in anti-depression in both in vivo and in vitro models.;A systematic approach was designed for the chemical standardization of KXS decoctions. By using rapid resolution liquid chromatography (RRLC) coupled with a diode-array detector (DAD) and an electrospray ionization triple quadrupole tandem mass spectrometry (QQQ-MS/MS), a rapid, simple, reliable and quantitative method to simultaneously determine the herb-derived chemical markers in different ratios of KXS was successfully developed. The eight marker chemicals, including ginsenoside Rb1, Rd, Re, Rg1 derived from Ginseng Radix et Rhizoma, 3',6-di-O-sinapoyl sucrose ester derived from Polygalae Radix, α-asarone, β-asarone derived from Acori Tatarinowii Rhizoma and pachymic acid derived from Poria, were used to standardize KXS extracts. The amounts of all marker chemicals, soluble in water, in the formulae were higher than that of single herb. K-652 showed the highest concentration of asarone (both α and β) and pachymic acid, while K-984 contained larger amount of ginsenosides and 3',6-di-O-sinapoyl-sucrose ester. These results suggested that a combination of herbs in KXS enhanced the chemicals being extracted in the decoctions. The established preparation method was able to ensure the quality of KXS in the following experiments.;The anti-depression effect of KXS was evaluated on a depressive animal model. K-652, the most popular formula used today, was evaluated on the depressive rats induced by the c&barbelow;hronic m&barbelow;ild s&barbelow;tress (CMS). Daily intra-gastric administration of a chemically standardized KXS for four weeks significantly alleviated the CMS-induced depressive symptoms displayed by enhanced sucrose consumption. The expressions of various molecular bio-markers in the depressive rat brains were altered by the treatment of KXS. These KXS-regulated brain bio-markers included: (i) the levels of dopamine, norepinephrine and serotonin; (ii) the transcript levels of proteins relating to neurotransmitter metabolism; and (iii) the transcript levels of neurotrophic factors and their receptors. The results suggested that the anti-depressant-like action of KXS might be mediated by an increase of neurotransmitters and expression of neurotrophic factors and its corresponding receptors in the brain.;A series of biological analyses were applied to evaluate the biological efficacy of KXS in cultured neurons and astrocytes. Upon treatment by n&barbelow;erve g&barbelow;rowth f&barbelow;actor (NGF), cultured PC12 cells were differentiated and neurite outgrowth was observed. The treatment of KXS alone did not induce neurite outgrowth in cultured PC12 cells; however, the co-treatment of low-dose NGF and KXS induced the neurite outgrowth. Thus, the differentiation effect of NGF could be potentiated by KXS, i.e. more neurite outgrowth was induced as compared to that of NGF alone. The expression of three types of neurofilament (NF) protein, NF200, NF160 and NF68 were enhanced in co-treating low-dose NGF and KXS.;KXS was further evaluated on synaptic protein expression in cortical and hippocampal neurons. After the treatment of KXS, the expression levels of s&barbelow;ynaptic v&barbelow;esicles protein (SV48, pre-synaptic marker) and the p&barbelow;ost s&barbelow;ynaptic d&barbelow;ensity protein (PSD95, post-synaptic marker) were enhanced dramatically, and K-984 showed better effect, which implied that KXS might be beneficial for synaptogenesis. In cultured astrocytes, the treatment of KXS could significantly enhance the expression and secretion of neurotrophic factors in cultured astrocytes, e.g. NGF, BDNF and GDNF, which implied that KXS might be helpful for the growth of neuron.;Taken together, the current results indicated that KXS might exert its anti-depressant function in animals by modulating neurotransmitter, inducing neuron differentiation, modulating the synaptogenesis and enhancing neurothrophic factor expression. |
