Abstract:

Objective To discuss the effect of knockdown of receptor-interacting protein kinase 3 （RIP3） on autophagy， pyroptosis， and ferroptosis in the human renal tubular epithelial HK2 cells under hypoxia/reoxygenation （H/R） conditions. Methods The lentiviral interference vector plasmid shRIP3 and negative control lentiviral interference vector plasmid shNC were transfected into the HK2 cells and the HK cells were divided into shRIP3 group and shNC group， and the normal cultured untransfected HK2 cells were regarded as blank group. After 48 h of transfection， real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to verify the lentiviral transfection efficiencies. The HK2 cells were divided into control group， H/R group， shNC+H/R group， and shRIP3+H/R group. CCK-8 method was used to detect the survival rates of the HK2 cells in various groups； immunofluorescence staining was used to detect the fluorescence intensities of light chain 3 （LC3B） and NLR family pyrin domain-containing 3 （NLRP3） proteins in the cells in various groups； Western blotting method was used to detect the expression levels of LC3Ⅱ， LC3Ⅰ， Beclin1， Caspase-1， Gasdermin D （GSDMD）， interleukin （IL）-1β， and IL-18 proteins in the HK2 cells in various groups； Kits were used to detect the ferri ion（Fe²⁺） levels in the cells in various groups. Results Compared with blank group and shNC group， the expression levels of RIP3 mRNA and protein in the HK2 cells in shRIP3 group were decreased （P<0.05）.The CCK-8 method results showed that compared with control group， the survival rate of the HK2 cells in H/R group was decreased （P<0.05）； compared with H/R group， the survival rate of the HK2 cells in shRIP3+H/R group was increased （P<0.05）. The immunofluorescence staining results showed that compared with control group， the fluorescence intensity of LC3B protein in the HK2 cells in H/R group was decreased （P<0.05）， and the fluorescence intensity of NLRP3 protein was increased （P<0.05）； compared with H/R group， the fluorescence intensity of LC3B protein in the HK2 cells in shRIP3+H/R group was increased （P<0.05）， and the fluorescence intensity of NLRP3 protein was decreased （P<0.05）. The Western blotting results showed that compared with control group， the ratio of LC3Ⅱ/LC3Ⅰ and the expression level of Beclin1 protein in the HK2 cells in H/R group were decreased （P<0.05）； compared with H/R group， the ratio of LC3Ⅱ/LC3Ⅰ and expression level of Beclin1 protein in the HK2 cells in shRIP3+H/R group were increased （P<0.05）； compared with control group， the expression levels of Caspase-1， GSDMD， IL-1β， and IL-18 proteins in the HK2 cells in H/R group were increased （P<0.05）； compared with H/R group， the expression levels of Caspase-1， GSDMD， IL-1β， and IL-18 proteins in the HK2 cells in shRIP3+H/R group were decreased （P<0.05）. Compared with control group， the expression levels of GPX4 and SLC7A11 proteins in the HK2 cells in H/R group were decreased （P<0.05）； compared with H/R group， the expression levels of GPX4 and SLC7A11 proteins in the HK2 cells in shRIP3+H/R group were increased （P<0.05）. Compared with control group， the Fe²⁺ level in the HK2 cells in H/R group was increased （P<0.05）； compared with H/R group， the Fe²⁺ level in the HK2 cells in shRIP3+H/R group was decreased （P<0.05）. Conclusion Targeted knockdown of RIP3 can induce the autophagy， inhibit the pyroptosis， and reduce the ferroptosis of the human renal tubular epithelial HK2 cells induced by H/R.

Key words: Human renal tubular epithelial HK2 cells, Hypoxia/reoxygenation, Receptor-interacting protein kinase 3, Autophagy, Pyroptosis, Ferroptosis