Fig 2: PS promotes the nuclear translocation of Nrf2 in HaCaT keratinocytes in H2O2-mediated oxidative stress. HaCaT keratinocytes were seeded at a density of 1 × 104 cells/mL and treated with the indicated concentrations of PS (0–400 µg/mL) in the presence and absence of 1000 µM H2O2 for 4 h. (A) Western blotting was performed using cytosolic and nuclear protein fractions against Nrf2 (61 kDa). ß-Actin and nucleolin (110 kDa) were used as the internal control for cytoplasmic and nuclear fractions, respectively. (B) HaCaT keratinocytes were cultured on 3% gelatin-coated cover slips, and the expression and nuclear translocation of Nrf2 were analyzed by immunofluorescence staining after treatment with PS (400 µg/mL) in the presence or absence of 1000 µM H2O2. In a parallel experiment, Western blotting analysis was performed to assess the expression of Keap1 (69 kDa) (C), and pPI3K (85 kDa) PI3K (85 kDa) and pAKT (62 kDa) and Akt (62 kDa) (D). ß-Actin was used as the internal control along with their respective total forms. Densitometry analysis of each protein was computed by using ImageJ. All data are averaged from three independent experiments and presented as mean ± the standard error of the median [*** p < 0.001 and * p < 0.05 vs. the untreated group (UT) and ### p < 0.001 and # p < 0.05 vs. the H2O2-treated group]. Scale bars = 100 µm.

Fig 3: The in vivo verification of lncRNA 74.1 inhibition of renal fibrosis by inhibition of miR-324-3p/NRG1-mediated PI3K/AKT signaling pathway. (A) lncRNA 74.1 expression in mouse renal tissues determined using RT-qPCR; (B) miR-324-3p expression in mouse renal tissues determined using RT-qPCR; (C) NRG1 expression in mouse renal tissues determined using RT-qPCR; (D) protein expressions of PI3K, p-PI3K, AKT, and p-AKT in mouse renal tissues by Western blot; (E) protein expressions of ATG5, ATG7, and LC3II/I in mouse renal tissues by Western blot; (F) TEM observation of autophagosomes in mouse renal tissues with different treatments; (G) proportion of LC3 positive cells in mouse renal tissues with different treatments by immunofluorescence assay; (H) protein level of fibrosis markers in in mouse renal tissues by Western blot. *p < 0.05 as compared with the sham group; #p < 0.05 as compared with the UUO + Lv-NC group. The measurement data were expressed as mean ± standard deviation. One-way ANOVA was used for data comparisons among multiple groups, followed by Tukey’s post hoc test. n = 6.

Fig 4: The mechanistic diagram illustrating the role of lncRNA 74.1/miR-324-3p/NRG1-mediated PI3K/AKT signaling pathway in renal fibrosis. In renal fibrosis, lncRNA 74.1 expressed poorly and miR-324-3p expressed highly. LncRNA 74.1 could bind to miR-324-3p and negatively regulate its expression, which further promoted the NRG1 expression and inactivated the PI3K/AKT signaling pathway, thereby inducing cellular autophagy, and inhibiting renal fibrosis.

Fig 5: LncRNA 74.1 inactivates the PI3K/AKT signaling pathway to promote autophagy and inhibit renal fibrosis. (A) Western blot results of PI3K, p-PI3K, AKT, and p-AKT expressions in HK-2 cells with different treatments; (B) RT-qPCR results of the expressions of lncRNA 74.1, miR-324-3p, and NRG1 mRNA in cells with different treatments; (C) Western blot results of NRG1, PI3K, p-PI3K, AKT, and p-AKT expressions in HK-2 cells with different treatments; (D) level of autophagy-related proteins by Western blot; (E) TEM observation of autophagosomes in HK-2 cells with different treatments; (F) proportion of LC3 positive cells in the HK-2 cells with different treatments by immunofluorescence assay; (G) protein levels of fibrosis markers in HK-2 cells with different treatments by Western blot; *p < 0.05 as compared with oe-NC + sh-NC or oe-NC + DMSO; #p < 0.05 as compared with oe-lncRNA 74.1 + sh-NC or oe-lncRNA 74.1 + DMSO; &p < 0.05 as compared with si-lncRNA74.1 + sh-NC or si-lncRNA74.1 + DMSO. The measurement data were expressed as mean ± standard deviation. One-way ANOVA was used for data comparisons among multiple groups, followed by Tukey’s post hoc test. The cell experiments were repeated three times independently.