Preventive carnosine supplementation attenuates painful diabetic neuropathy via restoring mitochondrial function and suppressing inflammasome activation

Figure 1 The rat model of painful diabetic neuropathy established through high-fat feeding combined with low-dose streptozotocin injection. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs the control group; two-way analysis of variance with Sidak’s post-hoc test for Figure 1A and B; Unpaired t-tests, two-tailed for Figure 1C, I, K-M; Linear mixed-effects models were applied for two-group comparisons of data in panels F-H, with animal ID set as a random intercept to account for clustered observations within individual rats; Figure 1A-C: n = 11; Figure 1D-H: n = 3 rats per group, with at least 50 mitochondria counted in 3 random fields per sample. For Figure 1F and G, each field was the statistical unit; for panel H, each individual mitochondrion was the statistical unit for mitochondrial length quantification; I: n = 8; J-M: n = 6. A: Paw withdrawal threshold in painful diabetic neuropathy (PDN) rats; B: Paw withdrawal latency in PDN rats; C: Motor nerve conduction velocity and sensory nerve conduction velocity in PDN rats; D: Ultrastructural analysis of nerve fibers in the dorsal root ganglion (DRG) of PDN rats. Scale bar = 2 μm; E: Examination of mitochondrial structure in the DRG of PDN rats. Scale bar = 500 nm; F: Measurement of the number of mitochondria per field; G: Proportion of abnormal mitochondria to total mitochondria; H: Measurement of mitochondrial length; I: Assessment of mitochondrial membrane potential in the DRG of PDN rats, in which quantitative data were acquired by a fluorescence microplate reader; J-M: Western blotting showing the expression level of proteins associated with mitochondrial fission protein dynamin-related protein 1 and fusion proteins MFN1/MFN2, including representative electrophoresis bands and relative expression levels. PDN: Painful diabetic neuropathy; MNCV: Motor nerve conduction velocity; SNCV: Sensory nerve conduction velocity; STZ: Streptozotocin; Drp1: Dynamin-related protein 1.

Figure 2 Dorsal root ganglia metabolomic profiling and correlation analyses between carnosine levels and behavioral pain indices, including paw withdrawal threshold and paw withdrawal latency. A: Principal component analysis; green represents quality control, blue indicates the control group, and yellow represents painful diabetic neuropathy; B: Partial least squares discriminant analysis; green represents quality control, blue indicates the control group, and yellow represents painful diabetic neuropathy; C: Volcano plot of differential metabolites; orange indicates upregulated differential metabolites, blue indicates downregulated differential metabolites, and gray indicates metabolites detected but not significantly different; D: Kyoto Encyclopedia of Genes and Genomes topological analysis, the color gradient represents the significance of pathways arranged by P value (Y-axis, darker red indicates smaller P values), while the size of circles represents pathway impact values (X-axis, larger circles indicate higher pathway impact value scores); pathway enrichment was analyzed using a hypergeometric test, and P values were adjusted for multiple comparisons using the Benjamini-Hochberg method to control the false discovery rate (corrected P < 0.05); E and F: Correlation analysis between carnosine levels and behavioral pain indices, including paw withdrawal threshold and paw withdrawal latency. n = 5 animals per group. QC: Quality control; PDN: Painful diabetic neuropathy; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 3 The impact of intraperitoneal injection of carnosine on behavioral, electrophysiological, ultrastructural, and neuronal functional abnormalities in painful diabetic neuropathy rats. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs the control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs the painful diabetic neuropathy group. Two-way analysis of variance with Sidak’s multiple comparisons test for Figure 3B-E. One-way analysis of variance with Tukey’s multiple comparisons test for Figure 3F, H, and N; Linear mixed-effects models were used for statistical analyses of data shown in panels Figure 3K-M, with animal ID designated as the random intercept to account for clustered microscopic observations nested within individual rats. Bonferroni-adjusted pairwise comparisons were performed for intergroup comparisons across the three experimental groups. Figure 3B-F: n = 11; Figure 3G and H: n = 3; Figure 3I-M: n = 3 rats per group, with at least 50 mitochondria counted in 3 random fields per sample. For Figure 3K and L, each field was the statistical unit; for Figure 3M, each individual mitochondrion was the statistical unit for mitochondrial length quantification; Figure 3N: n = 8. A: Flowchart of the experiments; B: Changes in body weight after intraperitoneal carnosine injection; C: Changes in blood glucose level following intraperitoneal carnosine injection; D: Paw withdrawal threshold in rats post-intraperitoneal carnosine injection; E: Paw withdrawal latency in rats post-intraperitoneal carnosine injection; F: Motor nerve conduction velocity and sensory nerve conduction velocity in rats following intraperitoneal carnosine injection; G and H: Results of whole-cell patch-clamp recordings at week 3 after streptozotocin injection; G: Representative traces of neuronal action potential; H: Statistical analysis of the action potential number in dorsal root ganglia (DRG) neurons of rats (n = 3); I: Ultrastructural changes of the DRG post-intraperitoneal carnosine injection. Scale bar = 2 μm; J: Alterations in mitochondrial structure in the DRG post-intraperitoneal carnosine injection. Scale bar = 500 nm; K: The number of mitochondria per field; L: Proportion of abnormal mitochondria relative to the total mitochondrial count; M: Mitochondrial length; N: Changes in mitochondrial membrane potential in the DRG following intraperitoneal carnosine injection. PDN: Painful diabetic neuropathy; MNCV: Motor nerve conduction velocity; SNCV: Sensory nerve conduction velocity; STZ: Streptozotocin.

Figure 4 Impact of intraperitoneal carnosine injection on mitochondrial fission and NOD-like receptor pyrin domain-containing protein 3 inflammasome activation in rats with painful diabetic neuropathy. ^cP < 0.001 vs the control group; ^dP < 0.05, ^fP < 0.001 vs the painful diabetic neuropathy (PDN) group; Linear mixed-effects models were used for the analyses in Figure 4B and D, with animal ID included as a random intercept to account for neurons nested within individual rats. Bonferroni-adjusted pairwise comparisons were performed for post hoc analyses. One-way analysis of variance followed by Tukey’s multiple comparisons test was used for Figure 4F-K. For Figure 4A-D, n = 3 rats per group. Multiple image fields and neurons were analyzed for each animal. For Figure 4E-K, n = 6 rats per group. A: Representative immunofluorescence images of dynamin-related protein 1 and neuronal marker neuronal nuclear antigen in the dorsal root ganglia (DRG) of control rats, rats with PDN, and carnosine-treated PDN rats. Scale bar = 20 μm; B: Variations in immunofluorescence intensity of dynamin-related protein 1 in DRG neurons among control, PDN, and carnosine-treated PDN rats; C: Representative immunofluorescence images of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and neuronal marker neuronal nuclear antigen in the DRG of control, PDN, and carnosine-treated PDN rats. Scale bar = 20 μm; D: Changes in immunofluorescence intensity of NLRP3 in DRG neurons among control, PDN, and carnosine-treated PDN rats; E-K: Western blotting indicating the expression levels of proteins associated with mitochondrial fission and NLRP3 inflammasome pathway, including representative electrophoresis bands and relative expression levels. NLRP3: NOD-like receptor pyrin domain-containing protein 3; Drp1: Dynamin-related protein 1; PDN: Painful diabetic neuropathy; DAPI: 4’,6-diamidino-2-phenylindole; NeuN: Neuronal nuclear antigen; IL: Interleukin.

Figure 5 The screening of optimal carnosine concentration in PC12 cells. ^bP < 0.01, ^cP < 0.001 vs the control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs the high-glucose (HG) group. One-way analysis of variance with Tukey’s multiple comparisons test for Figure 5A-C, E and F. For Figure 5A-C, n = 3; for Figure 5D-F, n = 4. A: Cell counting kit-8 (CCK-8) assay of viability in PC12 cells treated with HG and mannitol; B: CCK-8 assay of viability in PC12 cells treated with various concentrations of carnosine in a standard culture medium; C: CCK-8 assay of viability in PC12 cells treated with different concentrations of carnosine in a HG culture medium; D-F: Western blotting indicating changes in the expression levels of mitochondrial fission protein dynamin-related protein 1 and NOD-like receptor pyrin domain-containing protein 3 inflammasome in PC12 cells treated with various concentrations of carnosine in a HG culture medium. NLRP3: NOD-like receptor pyrin domain-containing protein 3; Drp1: Dynamin-related protein 1; HG: High-glucose.

Figure 6 Effects of carnosine intervention on mitochondrial membrane potential, mitochondrial fission, and NOD-like receptor pyrin domain-containing protein 3 inflammasome activation in PC12 cells. ^cP < 0.001 vs the control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs the high-glucose group. One-way analysis of variance with Tukey’s multiple comparisons test for Figure 6B, D-I. For Figure 6A and B, n = 5; for Figure 6C-I, n = 4. A: Representative fluorescence images of JC-1 monomers/aggregates in PC12 cells. Scale bar = 20 μm; B: Mitochondrial membrane potential level in PC12 cells; C-H: Impact on the expression levels of proteins related to mitochondrial fission and NOD-like receptor pyrin domain-containing protein 3 inflammasome activation in PC12 cells, including representative electrophoresis bands and relative expression levels; I: Enzyme-linked immunosorbent assay showing the effect on interleukin-1β expression level in PC12 cells. NLRP3: NOD-like receptor pyrin domain-containing protein 3; Drp1: Dynamin-related protein 1; HG: High-glucose; IL-1β: Interleukin-1β.

Figure 7 dynamin-related protein 1 knockdown inhibits high glucose-induced NOD-like receptor pyrin domain-containing protein 3 inflammasome activation, whereas dynamin-related protein 1 overexpression attenuates the inhibitory effect of carnosine in PC12 cells. For Figure 7A-F, ^bP < 0.01 vs the control group; ^cP < 0.001 vs the control group; ^dP < 0.05 vs the high-glucose (HG) group; ^fP < 0.001 vs the HG group. For panels H-O, ^gP < 0.05 vs the vector control group, ^hP < 0.01 compared with the vector control group, ⁱP < 0.001 vs the vector control group; ^jP < 0.05 vs the vector HG group, ^kP < 0.01 vs the vector HG group, ^lP < 0.001 vs the vector HG group; ^mP < 0.01 vs vector carnosine group, ⁿP < 0.001 vs vector carnosine group. One-way analysis of variance with Tukey’s multiple comparisons test for Figure 7B-F, H, J-O. For Figure 7A-F, I-O, n = 4, G-H, n = 5. A-E: Western blotting of mitochondrial fission and NOD-like receptor pyrin domain-containing protein 3 inflammasome activation-related protein expression following lentivirus-short hairpin RNA targeting dynamin-related protein 1 knockdown, including representative electrophoresis bands and relative expression levels; F: Enzyme-linked immunosorbent assay showing interleukin-1β expression level; G: Representative fluorescence images of JC-1 monomers/aggregates in PC12 cells. Scale bar = 20 μm; H: Mitochondrial membrane potential level in PC12 cells; I-N: Western blotting of mitochondrial fission and NOD-like receptor pyrin domain-containing protein 3 inflammasome activation-related protein expression after lentivirus-dynamin-related protein 1 overexpression, including representative electrophoresis bands and relative expression levels; O: Enzyme-li O: Enzyme-linked immunosorbent assay indicating interleukin-1β expression level in PC12 cells. NLRP3: NOD-like receptor pyrin domain-containing protein 3; Drp1: Dynamin-related protein 1; HG: High-glucose; IL-1β: Interleukin-1β; LV-shDrp1: Lentivirus-short hairpin RNA targeting dynamin-related protein 1.