Fig 2: Mechanism diagram. STZ and HG treatment induced high expression of X inactive specific transcript (XIST); silencing XIST promoted the targeted inhibition of miR-15b-5p on toll like receptor 4 (TLR4) by upregulating miR-15b-5p, thereby inhibiting TLR4, and ultimately alleviating the renal injury of diabetic nephropathy by inhibiting NLRP3/Caspase-1-mediated RTEC pyroptosis. mRNA: messenger RNA; DN: Diabetic nephropathy; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D.

Fig 3: Toll like receptor 4 overexpression in X inactive specific transcript-silenced cells causes increased pyroptosis. In HG-treated HK2 cells, X inactive specific transcript (XIST) was silenced and toll like receptor 4 (TLR4) was overexpressed. A and B: The expression of TLR4 levels detected by qRT-PCR and Western blot; C: Enzyme-linked immunosorbent assay detected the expression of IL-1ß and IL-18; D: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; E: Cell viability; F: LDH activity. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (A/B/E/F) or two-way ANOVA (C/D) and Tukey's multiple comparisons test; aP < 0.05. XIST: X inactive specific transcript; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LDH: Lactate dehydrogenase; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D.

Fig 4: Inhibition of miR-15b-5p in X inactive specific transcript -silenced HK2 cells activates renal tubular epithelial cell pyroptosis. In HG-treated HK2 cells, both X inactive specific transcript (XIST) and miR-15b-5p were silenced. A: qRT-PCR verified the transfection efficiency of miR-15b-5p inhibitor; B: Enzyme-linked immunosorbent assay detected the expression of IL-1ß and IL-18; C: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; D: Cell viability; E: Lactate dehydrogenase activity. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (A/D-E) or two-way ANOVA (B/C) and Tukey's multiple comparisons test; aP < 0.05. LDH: Lactate dehydrogenase; XIST: X inactive specific transcript; si: Small interfering RNA; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D.

Fig 5: Silencing lncRNA X inactive specific transcript can inhibit renal tubular epithelial cell pyroptosis in diabetic nephropathy rats. After establishing diabetic nephropathy model rats, lentivirus-LV sh- X inactive specific transcript was injected into the tail vein. A: Enzyme-linked immunosorbent assay detected IL-1ß and IL-18 expressions; B: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; C: Immunofluorescence tested the expression of Caspase-1; scale bar: 25 µm; N = 8/group. The data were described as mean ± SD and analyzed by one-way ANOVA and Tukey's multiple comparisons test; aP < 0.05. DN: Diabetic nephropathy; LV: Lentivirus; XIST: X inactive specific transcript; sh: Short hairpin RNA; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D; DAPI: 4',6-diamidino-2-phenylindole.