Protective Effects Of Quercetin, Hirudin, Cinnamaldehyde And Combination Of Them On Oxidative Stress And Apoptosis In Experimental Diabetic Neuropathy

[Objective]To investigate protective effects of quercetin, hirudin and cinnamaldehyde on oxidative stress and apoptosis in experimental diabetic neuropathy at the cellular and molecular level.[Methods]①DRGn are harvested from embryonic day15SD rats, dissociated in0.25%trypsin, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27minus AO additives and nerve growth factor. The immunofluorescence method with anti-MAP-2was used to identify the purity of the DRGn.②DRGn were divided into different groups, cultured with45mM glucose, different concentration of quercetin(Q), hirudin(H) and cinnamaldehyde(C) and ALA group. MTT assay was used to determine the optimal concentration and time point of the monomers. Then based on the results of the monomers, the MTT assay was used to determine the optimal combination of the monomers (QH, QC, CH, QCH)④:Flow cytometry assay was used to test the level of ROS; western blot and qRT-PCR was used to test the expression of Nrf-2、HO-1、NF-κB (P65)、IκB-α、 pIκB-α、IL-6TNF-αand Caspase-3; TUNEL assay was used to test the apoptosis rate of the different group.[Results]1. The effect of quercetin, hirudin and cinnamaldehyde on DRGn in vitro①MTT assay:After a24-h incubation under group-specified experimental conditions, cell viability levels in groups of Q, H or C with different doses, were much higher than the HG group (P<0.05or P<0.01). Q, H and C attenuated high glucose-induced decreases in cell viability in a dose-dependent manner.②Apoptosis:TUNEL analysis showed that the percentages of apoptotic cells were increased in groups exposed to high glucose. The addition of Q, H or C with different doses reduced the frequency of apoptotic DRG neurons in high glucose conditions significantly (P<0.01) and these inhibitory effects were dose dependent (P<0.01)③Oxidative stress:Measurement of intracellular hydrogen peroxide levels with the DCFH-DA assay revealed that HG produced a marked increase in intracellular ROS levels, compared to that seen in control cells. Q, H and C blocked this increase in intracellular ROS in a dose-dependent manner (P<0.01)④Effects of Q and C on NF-κB, IκB, p-IκB, IL-6and TNF-a levels:the induction of inflammatory mediators under HG exposure was paralleled by upregulation of expression of NF-κB and IκB, as well as phosphorylation of IκB. Western blot experiment revealed that levels of NF-κB, IκB, p-IκB, IL-6and TNF-a protein were higher in cells in the HG condition than in the control group. Compared of the HG group, Q-treated cells and C treated cells had decreased levels of those proteins (P <0.05or P<0.01), and this effect was dose dependent. Consistent with these Western blot protein data, our qRT-PCR experiment also showed reduced levels of NF-κB, IL-6and TNF-a mRNA in the Q-treated groups.⑤Effects of Q and H on the Nrf-2and HO-1expression:after24h, Nrf-2expression was decreased in the HG group (P<0.05or P<0.01), but increased in the Q or H groups, compared with the control group (P<0.01). HO-1was decreased in the HG group compared with controls. Treatment with Q or H significantly attenuated HG effects on HO-1protein expression. Indeed, the addition of Q normalized levels of Nrf-2and HO-1under HG conditions in a dose-dependent manner. Similar patterns of Nrf-2and HO-1mRNA transcription were detected across the different groups of cells.⑥Caspase-3:addition of Q, C or H in the media significantly reduced the Caspase-3cleavage in a dose-dependent manner (P<0.05or P<0.01), although levels of intact Caspase-3protein remained stable.2. The effect of quercetin, hirudin and cinnamaldehyde combination.①MTT assay:After a24-h incubation under group-specified experimental conditions, The cell viability levels in QCH combination groups were much higher than the other groups (P<0.01). There is no significant difference between QCH group and ALA group (P>0.05). Combination effect was better than the single action (P<0.01or P<0.05) ②Apoptosis:TUNEL analysis showed that the treatment groups all reduced the frequency of apoptotic DRG neurons in HG conditions significantly (P<0.01) and QCH had the lowest percentage similar to ALA group.③Oxidative stress:The treated groups were all decreased the level of ROS (P<0.01). There was no significant difference between CON group and QCH group. QCH group was much lower than the other groups, except ALA groups (P<0.01)④Effects on NF-κB, IκB, p-IκB, IL-6and TNF-α:Western blot experiments revealed that levels of NF-κB, IκB, p-IκB, IL-6and TNF-a proteins in treatment groups were lower than the HG group (P<0.01or P<0.05). Compared with ALA group, the expression of these proteins in monomer groups were lower, except the H3group (P<0.01or P<0.05). QCH group was lowest among the treatment groups. Our qRT-PCR experiment also showed similar results.⑤Effects on the Nrf-2and HO-1expression:After24h, compared with the HG group, Nrf-2and HO-1expressions were increased in the almost all the treatment groups, except C3group (P<0.01or P<0.05). There was no significant difference between QCH group and ALA group, QCH group was higher than the other treatment groups (P<0.01or P<0.05).qRT-PCR experiment also showed the similar results.⑥Caspase-3:DRGn with treatment groups’cleavage Caspase-3significantly reduced (P<0.01or P<0.05). QCH had the lowest percentage similar to ALA group. qRT-PCR experiment also showed the similar results.[Conclusion]①High glucose remarkably inhibited the cell viability, increased apoptosis percentage. The levels of ROS, NF-κB (P65), TNF-a, IL-6and Caspase-3in cultured in high glucose medium significantly increased compared to control group, but Nrf-2and HO-1levels significantly reduced.②Quercetin, hirudin and cinnamaldehyde separately significantly enhanced the cell viability and reduced apoptosis percentage of DRGn cultured in high glucose.③Quercetin, cinnamaldehyde and high dose of hirudin treatments significantly ameliorated the alteration in ROS, NF-ΚB (P65), TNF-a, IL-6, Caspase-3and the apoptosis percentage of DRGn. Meanwhile, the quercetin and hirudin could enhance the level of Nrf-2/HO-1. All the three monomers could downregulate the Caspase-3cleavage. ④The combinations of quercetin, hirudin and cinnamaldehyde significantly enhanced the cell viability and reduced apoptosis percentage of DRGn cultured in high glucose. The effect of QCH was better than QH、QC、CH group.⑤The combinations’ effects were better than the monomers’ single action, and the suppression of NF-κB pathway and Caspase-3level in QCH group is much higher than ALA group, but there is no significant difference between them in Nrf-2/HO-1pathway⑥These results suggest that quercetin, hirudin and cinnamaldehyde antagonized the oxidative stress, activation of the Nrf-2/HO-1pathway, suppression the NF-κB pathway and inhibition the apoptosis of DRGn cultured in high glucose medium.