3.1 TPC and TFC of CE
As shown in Fig. 1 (d) and (e), the standard curve equation of gallic acid was y = 0.0025x-0.0699, R2 = 0.9941, and the standard curve equation of rutin was y = 0.0016x-0.027, R2 = 0.9996. The TPC in CE was 57.37mg/g and the TFC was 41.56 mg/g.
3.2 Effect of NP administration on the viability of Hep G2 cells
As shown in Fig. 2(a), after using different concentrations of NP (3.125, 6.25, 12.5, 25, 50, 100, 200 µM) on Hep G2 cells for 24 h, cell viability was not significantly different compared with the control group at concentrations of 3.125 ~ 50µM; cell viability was reduced to 13.33% and 2.04% at concentrations of 100µM and 200µM, with highly significant differences compared with the control group (P < 0.01). To select the appropriate NP concentration, Hep G2 cells were treated with 40, 50, 60, 70, 80, 90 and 100 µM for 24 h. The results are shown in Fig. 2(b) at concentrations of 40µM and 50 µM was not significantly different compared with the control group; at concentrations of 60 µM and 70 µM, cell viability decreased to 87.89% and 55.03%, respectively, with highly significant differences compared with the control group (P < 0.01). Subsequent experiments were selected at 0, 40, 50, 60 and 70 µM.
To investigate whether NP produced cytotoxicity to Hep G2 cells, GSH content and CAT activity, which are indicators of oxidative stress, and ATP content, which is an indicator of mitochondrial function, were measured. As shown in Fig. 2(c)(d), when NP was applied to Hep G2 cells for 24 h, GSH content showed a dose-dependent decrease, and the decrease was statistically significant at 50, 60 and 70µM (P < 0.01). Meanwhile, CAT vitality showed a trend of the first decrease and then increased, and the vitality dropped to the lowest at 60 µM (P < 0.01), but there was no significant difference between the CAT vitality of 60 µM and 70 µM (P > 0.05). As shown in Fig. 2(e), when NP was applied to Hep G2 cells for 24 h, the ATP content showed a tendency to increase and then decrease, and the decrease was statistically significant at 60µM and 70µM compared with the control group (P < 0.05, P < 0.01).
3.3 Effect of NP administration on Nrf2 pathway in Hep G2 cells
To investigate whether NP affects the classical antioxidant and detoxification Nrf2 pathway, Hep G2 cells were treated with different concentrations of NP for 24 h. As shown in Fig. 3(a)(b)(c), compared with the control group, the protein contents of Nrf2, HO-1 and NQO1 decreased in a dose-dependent manner; Nrf2 protein content decreased significantly at 40, 50, 60 and 70µM (P < 0.01), and down-regulated its downstream proteins HO-1 and NQO1 protein content. The results indicated that NP affects the expression of Nrf2 pathway-related proteins.
3.4 Effect of NP administration on MAPKs pathway in Hep G2 cells
The Nrf2 pathway may be activated by its upstream MAPKs pathway in addition to being directly activated by ROS. To explore whether NP affects the MAPKs pathway, Hep G2 cells were treated with different concentrations of NP for 24 h. As shown in Fig. 3(d)(e)(f), The expressions of p-ERK and p-P38 appeared to increase and then decrease while the total expressions of ERK and total P38 gradually decreased with increasing doses of NP. p-ERK/ERK ratio showed a gradual increase, and p-P38/P38 showed an increase and then a decrease. This indicated that NP activated p-ERK and p-P38 at 40–60 µM, and at 70 µM, NP inhibits p-ERK and p-P38 by suppressing total ERK and total P38; while p-JNK/JNK is activated only at 70 µM (P < 0.01). The following experiments used the NP concentration with 60 µM.
3.5 Intervention effect of CE on NP-induced cytotoxicity
To determine the concentration range of CE intervention in Hep G2, Hep G2 cells were treated with different concentrations of CE (0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mg/mL) for 24 h. The results of CCK-8 experiments showed that CE at low concentrations (0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mg/mL) had an activating effect on Hep G2 cell viability (P < 0.05) at low concentration (0.125 mg/mL), while an inhibitory dose-dependent impact (P < 0.01) was observed at high concentrations (4–16 mg/mL).
The results of CCK-8 experiments showed a reduction in cell viability in the NP-treated group (P < 0.01), indicating that the NP cell injury model was successfully constructed; cell viability was increased after the intervention with different concentrations (0.25 to 2 mg/mL)compared with the NP-treated group (P < 0.01). Therefore, 0.25, 0.5, 1 and 2 mg/mL were selected as CE intervention concentrations in the subsequent experiments.
To investigate whether CE improved Hep G2 cytotoxicity caused by NP, GSH content, CAT activity and ATP content were analyzed; as shown in Fig. 4, GSH content was downregulated in the NP group compared with the control group (P < 0.05), while GSH content was upregulated after CE intervention (P < 0.05, P < 0.01). Meanwhile, CAT viability decreased in the NP group compared with the control group (P < 0.01) and increased after CE intervention with 1 mg/mL and 2 mg/mL (P < 0.01); furthermore, ATP content decreased in the NP group compared with the control group (P < 0.01), and increased in a dose-dependent manner after CE intervention (P < 0.01). It was suggested that CE's intervention effect was on NP-induced cytotoxicity.
3.6 CE alleviated the cytotoxicity of NP via the Nrf2 pathway
To further study the protective mechanism of CE on NP-treated Hep G2 cells, the expression of Nrf2-related pathway proteins was assayed. Nrf2 and HO-1 were down-regulated in the NP group compared with the control group (P < 0.05, P < 0.01). At the same time, the content of Nrf2 increased and then decreased after CE intervention, and the expression of its downstream pathway protein HO-1 was upregulated. This suggested that CE intervention regulates the expression of Nrf2-related pathway proteins in NP-treated Hep G2 cells.
3.7 CE alleviated the cytotoxicity of NP via the ERK1/2 pathway and P38
To investigate whether CE affected the expressions of MAPKs pathway proteins which were the upstream regulators of Nrf2, CE was applied to NP-treated Hep G2 cells for 24 h. As shown in Fig. 5, CE at 0.25-1 mg/mL dose-dependently down-regulated the activations of p-ERK/ERK and p-P38/P38 by NP, while NP combined with 2 mg/mL CE upregulated the relative expression of p-ERK/ERK, p-P38/P38 proteins, suggesting that CE at 0.25 ~ 1 mg/mL had a downregulatory effect on NP-induced MAPKs activation, and therefore, 1 mg/mL was chosen for the following experiments.
3.8 CE affected Nrf2 pathway through ERK1/2 and P38 to alleviate the cytotoxicity of NP
Next, we verified the effect of CE on the activation of p-ERK/ERK due to NP. ERK1/2 is an essential upstream signaling molecule in the Nrf2/HO-1 pathway. As shown in Fig. 6(a), NP group had an activating effect on p-ERK/ERK, while CE intervention significantly decreased the expression of p-ERK/ERK and upregulated the expression of downstream pathway proteins Nrf2 and HO-1. Meanwhile, ERK1/2 inhibitor (PD98059) down-regulated p-ERK/ERK expression and upregulated downstream pathway protein Nrf2 and HO-1 expression in NP-treated Hep G2 cells. These results were consistent with the results after CE intervention in NP-treated Hep G2 cells, suggesting that CE might upregulate the expression of transcription factor Nrf2 and its downstream detoxification protein HO-1 by inhibiting NP-induced p-ERK/ERK expression in Hep G2 cells.
To verify the effect of CE on NP-induced activation of p-P38/P38, the P38 inhibitor (SB202190) was used on NP-treated Hep G2 cells. p38 is the same as ERK1/2, which is an important upstream signaling molecule in the Nrf2/HO-1 pathway. As shown in Fig. 6(b), the NP group had an activating effect on p-P38/P38, while CE intervention significantly reduced the expression of p-P38/P38 and upregulated the expression of downstream pathway proteins Nrf2 and HO-1. Meanwhile, SB202190 down-regulated the expression of p-P38/P38 and upregulated the expression of downstream pathway proteins Nrf2 and HO-1 in NP-treated Hep G2 cells. These results were also consistent with the results after CE intervention in NP-treated Hep G2 cells, suggesting that CE might upregulate the expression of transcription factor Nrf2 and its downstream detoxification protein HO-1 by inhibiting NP-induced expression of p-P38/P38 in Hep G2 cells.
To verify whether CE regulated NP-induced Hep G2 cell injury via Nrf2, Hep G2 cells co-interacted with CE and NP were intervened using the Nrf2 inhibitor ML385. As shown in Fig. 6(c), the NP group downregulated the expression of Nrf2 and thus HO-1 compared to the control group, and the expression of Nrf2 and HO-1 increased after intervention with CE; further addition of the Nrf2 inhibitor (ML385) resulted in a dose-dependent downregulation of Nrf2 and HO-1 expression compared to the CE and NP co-interaction group; subsequently, it was explored whether Nrf2 inhibition affected the expression of upstream proteins ERK1/2, P38, and the results showed that NP group activated the expression of p-ERK/ERK, p-P38/P38, and the expression of p-ERK/ERK, p-P38/P38 was significantly reduced after the intervention of CE; meanwhile, the addition of Nrf2 inhibitor (ML385) dose-dependently upregulated the expression of p-ERK/ERK, p-P38/P38 compared with the CE and NP co-interaction group.