Objective To elucidate the regulatory role and the underlying molecular mechanisms of serine/threonine protein kinase 1(AKT1)in the lens epithelium of patients with age-related cataract(ARC).Methods 1)Differentially expressed genes in ARC were screened using bioinformatics analysis(Genecard and GEO database GSE213546),and key genes were identified through functional enrichment analysis(KEGG and GO).2)An oxidative stress model of lens epithelial cells was established by treating HLE-B3 cells with 200 μmol/L H2O2 for 24 h.RT-qPCR and Western blot were performed to assess AKT1 gene and protein expression changes.3)Model cells were randomly divided into a si-NC group transfected with si-NC plasmids and 3 parallel si-AKT1 groups transfected with 3 types of si-AKT1 plasmids.A control+si-NC group was also set up,in which HLE-B3 cells not treated with H2O2 were transfected with si-NC empty plasmids.The AKT1 gene and protein expression levels in the si-NC and si-AKT1 groups were determined by RT-qPCR and Western blot.Two si-AKT1 parallel groups demonstrating the most significant changes in expression levels were selected for further experiments.The protein expression levels of AMPK and phosphorylated AMPK(p-AMPK)in the si-NC group,the two selected si-AKT1 parallel groups,and the control+si-NC group were determined by Western blot.A si-AKT1 parallel group demonstrating significant changes in p-AMPK/AMPK values was selected and treated with Acadesine(AICAR),an AMPK agonist,to verify the role of the AMPK pathway.Western blot was performed to determine Bcl-2 and Bax protein levels in the si-NC group,the control+si-NC group,and the si-AKT1 group before and after the administration of AICAR.Flow cytometry was performed to measure apoptosis and reactive oxygen species(ROS)levels,while ELISA kits were used to assess the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and reduced glutathione(GSH).Results 1)Through bioinformatics analysis,78 differentially expressed genes were identified,with AKT1 significantly upregulated in ARC samples(P<0.05)and enriched in the AMPK pathway.2)Compared with cells not treated with H2O2,AKT1 mRNA and protein expression increased in the oxidative stress model cells.3)The p-AMPK/AMPK ratio was higher in the si-NC group than that in the control+si-NC group.In contrast,AKT1 knockdown suppressed AMPK pathway activity,with all the si-AKT1 groups showing a significantly decreased p-AMPK/AMPK ratio compared to that of the si-NC group(P<0.05).Compared with the control+si-NC group,the si-NC group exhibited elevated ROS and MDA levels,increased apoptosis rate,reduced SOD and GSH levels,downregulated Bcl-2,and upregulated Bax(all P<0.05).Compared to those in the si-NC group,these indicators were improved in the si-AKT1 group(all P<0.05).However,compared with the findings before AICAR treatment,these effects were antagonized after AICAR treatment in the si-AKT1 group(all P<0.05).Conclusion The oxidative stress-related gene AKT1 may be a key pathogenic factor in cataract,and AKT1 induces oxidative stress and apoptosis in lens epithelial cells by modulating AMPK pathway activity.

Objective To observe the clinical efficacy of staphylococcal protein A immunoadsorption plus nonmyeloablative chemotherapy with CD34+ autologous peripheral blood stem cell transplantation in the treatment of refractory systemic lupus erythematosus (SLE). Methods Three patients with active SLE were enrolled into this study. All patients were diagnosed with lupus nephritis by renal biopsy and poorly responded to routine therapy. Before transplantation, patients were given 6 sessions of immunoadsorption apheresis using columns of staphylococcal protein A-silica with an interval of 3 days; each session processed 3 L plasma and a total of 18 L plasma was processed over the 6 treatments. Three days following the immunoadsorption apheresis, the mobilization of stem cells was realized by intravenous cyclophosphamide at a dose of 2 g per square meter of body surface area and subcutaneous recombinant human granulocyte colony-stimulating factor (G-CSF) at a dose of 5 g per kilogram of body weight per day for 5 days. Then, peripheral blood raonoclonal cells were obtained by CS-3000 Cell Separator, and passed through the Clini Macs CD34+ cell selection device, with the final concentration of CD34+ cells being 2.6×106, 2.1×106 and 2.4×106 per kilogram of body weight respectively, and that of CD3+ cells being 3×105, 2.1×105, and 2.0×105 per kilogram of body weight, respectively, in these three patients. The conditioning regimen consisted of oral fludarabine of 50 mg/d for 5 days plus intravenous pig anti-human thymocyte immunoglobulin (ATG) at a daily dose of 90 mg/kg for 5 days. After 72-hour treatment with ATG, the frozen stem cells were infused back to the patients. Clinical manifestations and lupus-correlated immune parameters were compared in patients at baseline and after transplantation. Results Following immunoadsorption apheresis, an obvious decrease was observed in the level of serum anti-dsDNA, antinuclear antibody and IgG antibodies, while an increase in the level of serum complement 3. All patients achieved the reconstruction of hemopoiesis 2-3 days after the transplantation. Also, an apparent clinical remission was achieved with the SLEDAI score being less than 3. Six months after the transplantation, serum anti-dsDNA and antinuclear antibodies as well as urine protein were undetectable, the level of complement 3 reached the normal range, and renal function was restored. Conclusions Staphylococcal protein A immunoadsorption plus nonmyeloablative CD34+ autologous peripheral blood stem cell transplantation are effective and safe for refractory SLE, but the long-term effect remains to be connfirmed by further studies.