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. 2020 Sep 28;23(10):101617. doi: 10.1016/j.isci.2020.101617

Figure 6.

Figure 6

Dfe Treatment Alleviated Apoptosis of DRG Neuron in DPN Mice Involving Regulation of Bcl-2 Family Proteins and Caspase-3 by Inhibiting Kv2.1 Channel

(A and B) Representative images of double immune-fluorescent staining showing Annexin V-FITC and PI labeled early and late stage of apoptosis of DRG neuron from (A) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (B) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe). Hoechst was used to mark nuclei. Scale bar: 250 μm.

(C) Quantitative analyses for (A, B). N = 3. STZ versus Control by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 27.24. ∗p < 0.05, ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 14.77. ∗∗p < 0.01.

(D and E) The paraffin sections of DRG tissue from (D) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (E) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe) were fluorescently stained for IHC assay. Neurons were located by NeuN and apoptotic cells were labeled by tunnel. Scale bar: 25 μm.

(F) Quantitative analyses for (D, E). N = 3. STZ versus Control by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 54.09. ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 41.48. ∗∗p < 0.01, ∗∗∗p < 0.001.

(G and I) Western blot results demonstrated that Kv2.1 inhibitor Dfe reduced apoptosis in DRG neuron from (G) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (I) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe) by regulating apoptosis-related proteins. T-ERK was used as loading control. N = 3.

(H and J) Quantitative analyses for (G, I). Bcl-xl (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 14.33. ##p < 0.01; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.535. #p < 0.05), Bcl-2 (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.205. #p < 0.05; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 15.61. #p < 0.05, ##p < 0.01), Bax (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 11.06. #p < 0.05. ##p < 0.01; db/db versus db/m by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 8.733. #p < 0.05), p-Bad (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.134. #p < 0.05; db/db versus db/m by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.357. #p < 0.05), Cleaved-caspase-3 (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.543. #p < 0.05; db/db versus db/m by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 20.9. ##p < 0.01).

(K and L) Western blot assays in DRG tissue from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe). DRG tissue from three mice per group was mixed and homogenized. Bcl-xl (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ###p < 0.001. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.186. ns), Bcl-2 (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 6.773. ns. #p < 0.05), Bax (STZ + AAV-NC versus Control by Student's t test: ∗p < 0.05. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 2.492. ns), p-Bad (STZ + AAV-NC versus Control by Student's t test: ∗p < 0.05. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ns. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.101. &p < 0.05, ns), Cleaved caspase-3 (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.272. &p < 0.05, ns). All data were presented as means ± SEM.