Abstract:

Objective To discuss the effect of sericin on the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/nuclear factor-κB （NF-κB） signaling pathway and apoptosis in the streptozotocin （STZ）-damaged INS-1 cells， and to clarify its mechanism. Methods The INS-1 cells were cultured with complete medium containing 0， 0.1， 0.3， 1.0， 3.0， and 10.0 μmol·L^-1 Akt1 inhibitor A-674563， 10 mmol·L^-1 STZ， and 600 mg·L^-1 sericin， and divided into 0， 0.1， 0.3， 1.0， 3.0， and 10.0 μmol·L^-1 A-674563 groups， and the control group （complete medium without drugs） was set up. Cell counting kit-8 （CCK-8） method was used to detect the survival rates of the INS-1 cells， and the half-maximal inhibitory concentration （IC₅₀） value was calculated to determine the optimal inhibitory concentration of A-674563， which was further verified by Western blotting method. The INS-1 cells were divided into normal control group （complete medium）， model group （10 mmol·L^-1 STZ+complete medium）， and low， medium， and high doses of sericin groups （10 mmol·L^-1 STZ+150 mg·L^-1 sericin+complete medium， 10 mmol·L^-1 STZ+300 mg·L^-1 sericin+complete medium， and 10 mmol·L^-1 STZ+600 mg·L^-1 sericin+complete medium）. CCK-8 method was used to detect the survival rates of the INS-1 cells in various groups to determine the optimal concentration of sericin. Additionally， the INS-1 cells were divided into normal control group （complete medium）， model group （10 mmol·L^-1 STZ+complete medium）， sericin group （10 mmol·L^-1 STZ+600 mg·L^-1 sericin + complete medium）， and A-674563 group （10 mmol·L^-1 STZ+600 mg·L^-1 sericin+0.3 μmol·L^-1 A-674563+complete medium）. Flow cytometry was used to detect the apoptotic rates of the INS-1 cells in various groups； real-time fluorescence quantitative PCR （RT-qPCR ） method was used to detect the expression levels of Akt1， NF-κB， tumor necrosis factor-α （TNF-α）， and interleukin-6 （IL-6） mRNA in the INS-1 cells in various groups； Western blotting method was used to detect the expression levels of phosphorylated Akt1 （p-Akt1） and NF-κB proteins in the INS-1 cells in various groups； enzyme linked immunosorbent assay（ELISA） method was used to detect the levels of TNF-α and IL-6 in the INS-1 cells in various groups. Results The survival rates of the INS-1 cells in control group was 100.00%±0.00%； in 0， 0.1， 0.3， 1.0， 3.0， and 10.0 μmol·L^-1 A-674563+10 mmol·L^-1 STZ+600 mg·L^-1 sericin+complete medium groups， which were 82.50%±2.28%， 69.47%±1.94%， 51.51%±1.74%， 38.94%±1.57%， 24.79%±1.14%， and 19.85%±1.03%， respectively. The IC?? value of A-674563 for INS-1 cells was 0.3 μmol·L^-1， and 0.3 μmol·L^-1 A-674563 was selected for subsequent experiments. Compared with 0 μmol·L^-1 A-674563， the expression level of p-Akt1 protein in the INS-1 cells after treated with 0.3 μmol·L^-1 A-674563+10 mmol·L^-1 STZ + 600 mg·L^-1 sericin+complete medium was significantly decreased （P<0.05）. The CCK-8 results showed that compared with normal control group， the survival rate of the INS-1 cells in model group was significantly decreased （P<0.05）； compared with model group， the survival rates of the INS-1 cells in low， medium， and high doses of sericin groups were significantly increased （P<0.05）； compared with low and medium doses of sericin groups， the survival rate of the INS-1 cells in high dose of sericin group was significantly increased （P<0.05）. Thus， 600 mg·L^-1 sericin was selected for subsequent experiments. The CCK-8 results showed that compared with normal control group， the survival rate of the INS-1 cells in model group was significantly decreased （P<0.05）； compared with model group， the survival rate of the INS-1 cells in sericin group was significantly increased （P<0.05）； compared with sericin group， the survival rate of the INS-1 cells in A-674563 group was significantly decreased （P<0.05）. The flow cytometry results showed that compared with normal control group， the apoptotic rate of the INS-1 cells in model group was significantly increased （P<0.05）； compared with model group， the apoptotic rate of the INS-1 cells in sericin group was significantly decreased （P<0.05）； compared with sericin group， the apoptotic rate of the INS-1 cells in A-674563 group was significantly increased （P<0.05）. The RT-qPCR results showed that compared with normal control group， the expression level of Akt1 mRNA in the INS-1 cells in model group was significantly decreased （P<0.05）； compared with model group， the expression levels of Akt1 mRNA in the INS-1 cells in low， medium， and high doses of sericin groups were significantly increased （P<0.05）； compared with low and medium doses of sericin groups， the expression level of Akt1 mRNA in the INS-1 cells in high dose of sericin group was significantly increased （P<0.05）. Compared with normal control group， the expression levels of NF-κB， TNF-α， and IL-6 mRNA in the INS-1 cells in model group were significantly increased （P<0.05）； compared with model group， the expression levels of NF-κB， TNF-α， and IL-6 mRNA in the INS-1 cells in sericin group were significantly decreased （P<0.05）； compared with sericin group， the expression level of NF-κB mRNA in the INS-1 cells in A-674563 group was significantly increased （P<0.05）. The Western blotting results showed that compared with normal control group， the expression level of p-Akt1 protein in the INS-1 cells in model group was significantly decreased （P<0.05）； compared with model group， the expression levels of p-Akt1 protein in the INS-1 cells in low， medium， and high doses of sericin groups were significantly increased （P<0.05）； compared with low and medium doses of sericin groups， the expression level of p-Akt1 protein in the INS-1 cells in high dose of sericin group was significantly increased （P<0.05）. Compared with normal control group， the expression level of NF-κB protein in the INS-1 cells in model group was significantly increased （P<0.05）； compared with model group， the expression level of NF-κB protein in the INS-1 cells in sericin group was significantly decreased （P<0.05）； compared with sericin group， the expression level of NF-κB protein in the INS-1 cells in A-674563 group was significantly increased （P<0.05）. The ELISA results showed that compared with normal control group， the levels of TNF-α and IL-6 in the INS-1 cells in model group were significantly increased （P<0.05）； compared with model group， the levels of TNF-α and IL-6 in the INS-1 cells in sericin group were significantly decreased （P<0.05）； compared with sericin group， the levels of TNF-α and IL-6 in the INS-1 cells in A-674563 group were significantly increased （P<0.05）. Conclusion Sericin alleviates the PI3K/Akt/NF-κB signaling pathway-mediated inflammatory response and apoptosis by targeting Akt1， exerting a protective effect against STZ-induced damage in INS-1 cells.

Key words: Sericin, INS-1 cells, Phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-κB signaling pathway, Inflammatory response, Apoptosis