1. Twenty percent OLVafter serum induced the most obvious inflammation and oxidative stress in A549 cells.
After 48 h of intervention with 10%, 20%, and 30% OLVbefore serum or OLVafter serum in A549 cells, the cell morphology was observed with a microscope in each group. As shown in Fig. 1A, when compared with the control group, in both the OLVbefore serum and OLVafter serum intervention groups, with the increase in serum concentration, the cell density per unit area gradually decreased, while the number of irregular cells gradually increased. Then, the expression of IL-6 at the protein and gene levels was detected by Western blot and qRT–PCR, respectively. The results indicated that 20% OLVafter serum could induce the highest level of IL-6 among all the groups, at both the protein and genetic levels, and produces the most obvious inflammatory response in A549 cells (Fig. 1B, 1C and 1D). In addition, the ROS assay showed similar results, which confirmed that 20% OLVafter serum could induce a significant oxidative stress response (Fig. 1E).
2. Pretreatment with 40 µg/ml curcumin in A549 cells is a suitable concentration to alleviate OLV serum-induced inflammation and oxidative stress.
A549 cells were pretreated with different concentrations of curcumin for 2 h, followed by 48 h of 20% OLVafter serum intervention. Then, the expression of IL-6 and the level of ROS were assessed. As shown in Fig. 2A, 2B and 2C, 20% OLVafter serum significantly increased the level of IL-6 at both the protein and gene levels. Compared with the control group, pretreatment with 40 µg/ml, 80 µg/ml and 160 µg/ml curcumin alone without serum intervention slightly increased IL-6 levels, but no significant difference was observed. Compared with the serum intervention group, pretreatment with curcumin at 40 µg/ml and 80 µg/ml in serum intervention cells reduced the protein level of IL-6 most significantly. In the ROS assay, the results indicated that 20% OLVafter serum could cause an obvious oxidative response. After pretreatment with curcumin, the production of ROS decreased significantly, and the reduction level of ROS in the three curcumin treatment groups was similar (Fig. 2D). Based on the above results, to minimize additional drug damage to cells, we chose the lower concentration (40 µg/ml) of curcumin for the following experiments.
3. Curcumin pretreatment reduced OLV serum-induced inflammation and oxidative stress and could be related to Prdx6.
Pretreatment with 40 µg/ml curcumin was used to intervene in the inflammation and oxidative stress induced by 20% OLVafter serum in A549 cells and to explore the effect of curcumin on the injury induced by serum collected from patients undergoing OLV in alveolar epithelial cells. We detected the protein expression of classic proinflammatory and anti-inflammatory cytokines, such as IL-6, IL-1β, TNF-α and IL-10, by Western blot. At the same time, the expression of MDA and SOD in the cell culture supernatant was quantified using an ELISA kit. As shown in Fig. 3A, 3B and 3C, serum from patients undergoing OLV’ stimulation produced obvious inflammatory and oxidative reactions, mainly manifested by an increase in expressions of IL-6, IL-1β, TNF-α and MDA and a decrease in expressions of IL-10 and SOD. Pretreatment with curcumin reversed these changes. Similarly, the results of the ROS assay also confirmed that curcumin could significantly reduce the increase in ROS content induced by OLV serum (Fig. 3D). Furthermore, we observed the expression of Prdx6 in each group by Western blot, qRT–PCR and immunofluorescence and found that OLV serum could significantly reduce the expression of Prdx6, while pretreatment with curcumin could reverse it (Fig. 3E, 3F and 3G).
4. Prdx6 in the protective effects of curcumin pretreatment on serum from OLV patient-induced injury and association with the NF-κB pathway.
To explore the potential mechanism of the protective effect of curcumin, we examined the proteins involved in the NF-κB pathway. After OLV serum intervention, the expression of P-IKKα/β and P-IKBα was increased significantly, while that of IKBα was decreased. When curcumin was used, the expression of P-IKKα/β and P-IKBα was significantly decreased, while that of IKBα was increased (Fig. 4A). To verify the relationship between Prdx6 and the NF-κB signaling pathway, we transfected A549 cells with Prdx6-siRNA or NC-siRNA. As shown in Fig. 4B and 4C, Prdx6-siRNA significantly reduced the expression of Prdx6 at both the protein and gene levels. Among them, Prdx6-siRNA 2 and siRNA 3 possessed the most obvious reduction effect. Therefore, we used Prdx6-siRNA 2 or siRNA 3 to transfect A549 cells. The transfected cells were pretreated with 40 µg/ml curcumin for 2 h, and an inflammation and oxidative stress injury model induced by 20% OLVafter serum was established. Compared with that in the NC-siRNA group, the expression of pro-inflammatory factors, including IL-6, IL-1β and TNF-α, in the Prdx6-siRNA group was significantly increased and anti-inflammatory IL-10 markedly reduced (Fig. 4D). ROS assays demonstrated that ROS generation in the Prdx6-siRNA group was significantly higher than that in the NC-siRNA group (Fig. 4E). Our ELISA results further confirmed that when compared with the NC-siRNA group, the expression of the pro-oxidative stress factor MDA significantly increased, while the expression of the antioxidative stress factor SOD obviously decreased in the Prdx6-siRNA group (Fig. 4F). Subsequently, we detected NF-κB pathway-related proteins in these two groups and found that, compared with the NC-siRNA group, the expression of P-IKKα/β and P-IKBα was significantly increased, while the expression of IKBα was slightly decreased in the Prdx6-siRNA group (Fig. 4G).
5. Prdx6 inhibits activation of the NF-κB signaling pathway by suppressing the nucleus translocation of P65 to participate in curcumin pretreatment on serum from OLV patient-induced inflammatory damage.
NC-siRNA- or Prdx6-siRNA-transfected A549 cells were pretreated with 40 µg/ml curcumin for 2 h, and an inflammation and oxidative stress injury model induced by 20% OLVafter serum was established. Immunofluorescence staining was performed, and the ProtLytic Nuclear and Cytoplasmic Protein Extraction kit was used to extract the cytoplasmic protein and nucleoprotein of the two groups to detect P65 expression in the treated cells. The results of the two detection methods in Fig. 5A and 5B showed that P65 was in both the cytoplasm and nucleus of curcumin-pretreated A549 cells. Additionally, compared to the NC-siRNA group, the expressions of P50 and P65 in the nucleus of the Prdx6-siRNA group were significantly higher, suggesting that Prdx6 suppressed P65 and P50 translocation into the nucleus to inhibit activation of the NF-κB signaling pathway. To further confirm the role of the NF-κB signaling pathway in the protective effect of curcumin pretreatment on serum from OLV patient-induced inflammatory damage, we performed a rescue assay using SC75741, which is a specific NF-κB inhibitor. As shown in Fig. 4D, SC75741 attenuated inflammatory damage induced by 20% OLVafter serum in A549 cells, as determined by inhibiting the expression of proinflammatory factors such as IL-6, IL-1β and TNF-α and increasing the levels of anti-inflammatory IL-10.
In summary, curcumin preconditioning exerted a protective effect on lung injury induced by serum from OLV patients in A549 cells by increasing the expression of Prdx6. On the one hand, Prdx6 can reduce the inflammatory response by suppressing the nuclear translocation of P65 to inhibit activation of the NF-κB signaling pathway, and on the other hand, Prdx6 can also reduce oxidative stress injury by increasing the activity of antioxidant enzymes and inhibiting the activity of pro-oxidation factors.