| 3,6 '- disinapoly sucrose?DISS? and Tenuifoliside A?TFSA? are two main active ingredients in Radix polygalae.Preliminary studies of our group have found that DISS has obvious antidepressant effect.It can change the behavioral index of depression mice by enhancing the function of 5-HT system.It also can regulate the HPA axis hormone levels in chronic stress rats and increase the expression of phosphorylated CREB in the hippocampus of depressed animals, and then improve the expression of BDNFmRNA and the downstream target protein of CREB. TFSA can reduce the effect of corticosterone on SH-SY5 Y cells by increasing the synthesis of DNA in newborn cells,TFSA can significantly increase the expression of phosphorylated CREB and brain derived neurotrophic factor in the hippocampus of depression animal induced by chronic stress and can regulate the function of HPA axis in rats with chronic stress,it has obvious anti-depressive effect. Further mechanism study has found,DISS plays a role of neural protection by activating the key target proteins of the TrkB/ERK and CaMK signaling pathway,and finally activate the phosphorylation of CREB, increase the expression of its downstream key proteins.And the neuroprotective effect of TFSA is related to the TrkB/ERK and TrkB/PI3 K signaling pathways. However, it is not clear whether the combination of DISS and TFSA has a synergistic effect. In this experiment,we will use a cell model of depression which is SH-SY5 Y cells damaged by glutamate, the suspension of mouse tailmodel and the forced swimming model, CCK-8 method and RT-PCR method, high performance liquid phase chromatography, Western blotting and immunohistochemistry methods to investigate the neuroprotective effects of DISS combination with TFSA in SH-SY5 Y cells and its mechanism, the experimental results are as follows:1. The combined index of DISS and TFSA is less than 0.7, according to the principle of the combined index method?CI? to determine the combination of DISS and TFSA have a synergistic effect.2. The results of flow cytometry showed that DISS and TFSA had a synergistic effect in resisting the damage of glutamate on SH-SY5 Y cells,and reducing the apoptosis of the cells.3. The contents of tNOS and iNOS were significantly increased in SH-SY5 Y cells induced by glutamate?P < 0.05?.DISS at the dose of75?M and TFSA at the dose of 25?M could significantly inhibit the increase of tNOS and iNOS in the cells induced by glutamate when given alone,and co-administration. And the inhibitory effect of the combination group was more obvious. The results suggest that the protective effects of DISS and TFSA may be related to the inhibition of NO production in SH-SY5 Y cells.4. The combination of DISS and TFSA can increase the expression of the key protein of CREB pathway in cells, but the high dose group was significantly more effective than the low dose group. TFSA at the doses of 75?M and 25?M can increase the expression of CRTC1,pCREB/CREB and BDNF in a degree, but the effect of the low dose on the protein expression was not significant. Compared with the single dose,the combination groups could significantly enhance the expression of CRTC1, pCREB/CREB and BDNF. At the same time, the dosage of DISS is particularly prominent in the combination of DISS and TFSA,the DISS+TFSA?150?M +50?M? and DISS+TFSA?150?M +25?M? were the best compatibility group of four groups.5. DISS at the does of 150?M and 75?M and TFSA at the does of 50?M can increase the expression of BDNF mRNA in SH-SY5 Y cells.Compared with the single drug group,the combination groups of DISS+TFSA?150?M+25?M? and DISS+TFSA?75?M+50?M? were significantly increased BDNF mRNA expression in SH-SY5 Y cells, and they had statistical significance.6. When using the antagonist of U0126 and LY294002,we found that the protective effect of DISS could be blocked by U0126, while LY294002 had no significant effect on its cell protective effect; The protective effect of TFSA cells could be inhibited by the antagonist LY294002;Compared with the single drug,the combination of DISS and TFSA was more obvious in the inhibition of glutamate induced cell damage.And the protective effect of DISS and TFSA in combination can obviously be blocked by the combination of U0126 and LY294002. Further protein immunoblotting results were found that U0126 and LY294002 could inhibit the expression of CRTC1 increased by DISS, but only U0126 could inhibit the increase of CRTC1 expression induced by TFSA; And the effect of DISS and TFSA on the increase of BDNF protein was inhibited by LY294002 and U0126 respectively.7. DISS(at the dose of 10-20 mg·kg-1) and TFSA(at the dose of 10-20mg·kg-1) can present a dose dependent trend to improve the behavioral parameters of the tail mice, and improve the content of NE?DA?5-HT in the hippocampus and the main protein on the CREB pathway, such as CRTC1?p CREB? BDNF, etc. Which were in agreement with previous experimental results. However, the combination of DISS and TFSA at the dose of 20 mg·kg-1 did not show a significant synergistic effect in improving the behavior. The low dose of DISS and TFSA at 5 mg·kg-1had no significant effect on the behavior of tail suspension mice.But the antidepressant effect of the combination DISS(at the dose of 5 mg·kg-1and 10mg·kg-1) with TFSA(at the dose of 5 mg·kg-1 and 10 mg·kg-1) wassignificantly more than the single dose of DISS and TFSA. It was suggested that DISS combinated with TFSA had synergistic effect in low and medium doses(5 mg·kg-1 and 10 mg·kg-1)?P<0.05?. Further protein immunoblotting results revealed, compared with the single drug group,the coadministration of DISS and TFSA at the doses of 5 mg·kg-1and 10 mg·kg-1 could significantly increase the expression of CRTC1?pCREB/CREB and BDNF in the hippocampus of the rat model of despair,and the results are consistent with the behavior.8. In forced swimming test in mice, the antidepressant effect of DISS(at the dose of 5 mg·kg-1 and 10mg·kg-1) combinated with TFSA(at the dose of 5 mg·kg-1 and 10 mg·kg-1) significantly exceeded the DISS and TFSA alone, which showed a significant antidepressant synergistic effect.Further immunohistochemical results showed that the coadministration of DISS and TFSA at the doses of 5 mg·kg-1 and 10 mg·kg-1 could significantly increase the expression of CRTC1 ? p CREB/CREB and BDNF in rat cortex and hippocampusIn conclusion, DISS combined with TFSA showed a better synergistic neuroprotective and anti-depressant effects in vitro and in vivo.When DISS combined with TFSA in certain dose and rate, they can partly start the downstream gene transcription of the MAPK and PI3 K pathways involved in CREBSer133 phosphorylation, and CTRC1 regulation CREBSer133 non phosphorylation, such as BDNF, The multiple targets can enhance the activity of the CREB-BDNF pathway,activate the neurotrophic pathway and gene expression related to neural differentiation? survival and protection, and play a synergistic role in the protective effect of nerve protection. But the exact molecular mechanism needs to be further studied. |
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