| BackgroundThe Chinese herbal medicine formula Tao Hong Si Wu decoction (THSWD) is traditionally used in China for cerebrovascular diseases. However, the molecular targets mechanisms of THSWD are largely unknown. In this study, we will first confirm the protective effect of THSWD on cerebral ischemic injury and then elucidate the underlying molecular mechanisms via the techniques of differential proteomics and molecular pharmacology. This work was supported by the TCM Scientific Program Grant (2012-2-87) from Chongqing Municipal Health Bureau Grant.ObjectiveThe objective of this study is to confirm the protective effect of THSWD on cerebral ischemic injury, identify the potential protein targets, characterize the key protein signaling pathway and finally elucidate the molecular mechanisms.Methods1 Protective effect of cerebral ischemic injury by THSWDTo determine the protective effect of THSWT on cerebral ischemic injured rat, the neurological function and infarct volume were determined in tMCAO rats. To determine the protective effect of THSWT on PC12 cells, the oxygen glucose deprivation-reoxygenation (OGD-Reoxy) model was used as the in vitro model and CCK-8 assay was applied to test the cell viability.2 Proteomics Identification of potential target proteins induced by THSWDAnalysis of target proteins induced by THSWD with differential proteomics: The two-dimensional gel electrophoresis-based proteomics was applied to investigate the differential protein profiles in PC 12 cells with or without the treatment of THSWD. The different proteins will be identified by MALDI-TOF mass spectrometry.Confirmation of the key target protein at the transcription level with quantitative PCR:To examine whether the target protein alterations observed by proteomic analysis correlate with the changes of those mRNAs at the transcription level, quantitative PCR was used to mRNA expression of those genes after THSWD treatment.Determination of the inducing effect of individual ingredients in THSWD on the key target proteins:Western blotting analysis was used to determine the inducing activity of the individual ingredients of THSWD on the key target protein.3 Determination of the role of the key protein pathway in the protective effect of THSWD on cerebral ischemic injuryWestern blotting was used to determine the expression of the key phase Ⅱ enzyme HO-1 activated by THSWD in vitro model. Then, Nrf2 nuclear translocation was characterized by Western blotting and immunocytochemical staining. Meanwhile, Western blotting and specific inhibitors were applied to determine the upstream protein kinases of Nrf2 pathway activated by THSWD. Finally, PI3K inhibitor and ARE decoy ODNs were used to determine the potential effect of PI3K/Akt and Nrf2 signaling pathway on THSWD’s protective effect.Results1 The protective effect of cerebral ischemic injury by Tao Hong Si Wu DecoctionI/R induced a significant decrease in neurological score, compared with rats in the sham group, indicating that the in vivo model was successfully established. Pretreatment with 0.5 g·kg-1·day-1,1.0 g·kg-1·day-1 or 1.5 g·kg-1·day-1 of THSWD significantly increased neurological score in I/R rats (P<0.01, P<0.001), indicating that THSWD exhibited protective effect on cerebral ischemic injury. Among them,1.0 g·kg-1·day-1 of THSWD showed the best protective effect. Compared with sham group, I/R caused significant infarct volume in rat brain, indicating the model was successfully established.THSWD in all groups (0.5g·kg-1·day-1,1.0g·kg-1·day-1 and 1.5 g·kg-1·day-1) signif icantly reduced the infarct volume compared that in the vehicle group, whereas 1.0 g·k g-1·day-1 of THSWD exhibited the largest effect. These results demonstrated that THS WD protected I/R injured rat brain.Compared with normal CTRL, THSWD did not show toxicity up to the concentration of 2mg·mL-after 24 h treatment, indicating THSWD within this range is safe for the subsequent protection study.. We then adapted an in vitro OGD-Reoxy procedure to mimic ischemic stroke. OGD-Reoxy induced significant cell injury compared with normal CTRL, indicating this model was successfully established. Notably, THSWD at concentrations ranging from 0.5 mg·mL-1 to 1.5 mg·mL-1 could significantly protect cells from OGD-Reoxy induced cell death (P<0.01), indicating that THSWD showed a protection against OGD-induced injury in PC12 cells.2 Proteomics Identification of potential target proteins induced by THSWDAnalysis of target proteins induced by THSWD with differential proteomics: Twenty-six proteins affected by THSWD were identified by MALDI-TOF mass spectrometry. Gene Ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions.Nrf2 mediated phase Ⅱ antioxidant enzymes is the key target proteins of THSWD:Six of them were found to be phase Ⅱ antioxidant enzymes, which regulated by Nrf2. Quantitative PCR further confirmed a dose-dependent induction of the six phase Ⅱ enzymes by THSWD at the transcription level.Synergistic effect may exist among the individual ingredients of THSWD on HO-linduction:PC12 cells were first treated with individual ingredients. HO-1 protein expression was detected by western blotting using the specific antibody. Notably, CX and CS were found to be very strong inducer for HO-1 expression (P<0.001). HH also significantly induced HO-1 expression (P<0.01). DG showed the weak HO-1 inducing potency (P<0.05), whereas the other ingredients showed no detectable induction of HO-1 expression (P>0.05).A step-wise deletion strategy was applied. THSWD significantly induced HO-1 expression (P<0.001). Deletion of CX or DG caused very large decrease in the level of HO-1 protein (P<0.001). Deletion of HH or CS also caused a large decrease but to a less extent (P<0.01). When TR or SD, which showed no effect on HO-1 induction previously, was deleted, the capability of the parent formula in inducing HO-1 expression also reduced (P<0.05). SWD, another formula used for improving women’s health and formed after deletion of TR and HH from THSWD, showed less potency on HO-1 induction compared with that of THSWD (P<0.05). Taken together, these results suggest that CX, DG, HH and CS are the main active ingredients for inducing HO-1 expression in THSWD. However, the capability of the whole formula in inducing HO-1 expression seems to contain the contribution of all individual ingredients and synergistic effect may exist among them.3 Determination of the role of the key protein pathway in the protective effect of THSWD on cerebral ischemic injuryIn cellular model, the results showed that THSWD induced the HO-1 expression in a concentration-(0.25 mg·mL-1,0.5 mg·mL-1and 1.0 mg·mL-1) and time-dependent (0-18 h) way and significantly promoted the nuclear translocation of Nrf2 proteins. Both 0.5 mg·ml-1 and 1.0 mg·ml-1 of THSWD significantly protected PC 12 cells against OGD-Reoxy induced cell death (P<0.01). The protective effect of 1.0 mg-ml"1 of THSWD significantly decreased in the presence of 20μM. LY294002 (P<0.05), whereas the protective effect of 0.5 mg-ml"1 of THSWD exhibited a decrease trend but without statistical significance in the presence of 20 μM LY294002. ARE decoy ODNs could inhibit the expression of Nrf2 driving phase Ⅱ enzymes. In this study, ARE decoy ODNs significantly reduced THSWD mediated protection against OGD-Reoxy (P<0.001). Although ARE mut ODNs also slightly reduced THSWD mediated protection, the reducing extent caused by ARE decoy ODNs significantly larger than that caused by ARE mut ODNs (P<0.01). It is thus clear that both LY294002 and ARE decoy ODNs significantly decreased THSWD-mediated protection against OGD-Reoxy, suggesting that THSWD ameliorate OGD-Reoxy induced injury via PBK/Akt and Nrf2 signaling pathway.Conclusion1. In this study, we confirmed the protection effect to ischemic brain injury by THSWD in vitro and in vivo models.2. Twenty-six target proteins were separated and identified by differential proteomics. And the phase II antioxidant enzymes regulated by Nrf2 may be the key target protein induced by THSWD. Synergistic effect may exist among the individual ingredients of THSWD on HO-1 induction.3. Finally, we characterized THSWD’s effect on PI3K/Akt and Nrf2 signaling pathway, and proved that it is the key molecular mechanisms (at least partially) responsible for the protective effect of THSWD. This study is valuable for providing a theoretical basis inmolecular mechanism for THSWD clinical application. |
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