Mechanism of anti-HuD autoantibody inducing enteric neuronal apoptosis of irritable bowel syndrome and its potential for targeted intervention

Figure 1 Indicators of the irritable bowel syndrome model established by passive transfer of a commercial anti-HuD autoantibody. A: Changes in body weight of the three groups of rats during the observation period; B: Changes in the number of fecal pellets of the three groups of rats during the observation period; C: Changes in the water content of feces of the three groups of rats during the observation period; D: Abdominal withdrawal reflex scores of three groups of rats under different pressures; E: Recorded pressure of three groups of rats during abdominal withdrawal reflex in which 3 represents the visceral pain threshold; F: Comparison of the intestinal transit rate of the three groups of rats; G: Representative image of the propelling distance of activated carbon powder in the intestine shown by the arrow. Data are expressed as mean ± SD. B-D were analyzed by two-way analysis of variance with Tukey’s multiple comparison test; E and F were analyzed by one-way analysis of variance with Tukey’s multiple comparison test. For A-F, n = 6 for the normal saline group and blank control group and n = 12 for the HuD autoantibody group. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001.

Figure 2 Anti-HuD autoantibody induced apoptosis of primary enteric neurons. A: Expression of apoptotic proteins detected by western blot; B: Morphological changes after 48 hours of antibody incubation. More apoptotic cells with vacant cytoplasm, partial atrophy, and reduced refraction were observed in the treated group. The yellow arrows indicate apoptotic neurons. Scale bar: 60 μm; C: Immunofluorescence detected the expression of cleaved caspase 3, and anti-PGP 9.5 indicated neurons; D: Immunofluorescence and terminal-deoxynucleotidyl transferase mediated nick end labeling staining detected nuclei cleavage. Anti-PGP 9.5 indicated neurons. The yellow arrows indicate apoptotic neurons. Scale bar: 20 μm; E: Statistical analysis of the relative protein expression of Bcl-2, Bax, and cleaved caspase 3; F: Statistical analysis of the percentage of apoptosis using cleaved caspase 3 immunostaining; G: Statistical analysis of the percentage of apoptosis using terminal-deoxynucleotidyl transferase mediated nick end labeling immunostaining. Data are expressed as mean ± SD; Student’s t-test was performed, n = 6 for each group. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling.

Figure 3 HuD protein counteracted the anti-HuD autoantibody to alleviate apoptosis in SH-SY5Y cells. A: Western blot analysis showing the effect of HuD plasmid transfection in counteracting HuD autoantibody-induced expression of apoptotic proteins in SH-SY5Y cells ; B: Terminal-deoxynucleotidyl transferase mediated nick end labeling showing the effect of HuD plasmid transfection antagonizing the HuD autoantibody on nuclei cleavage in SH-SY5Y. Neurons are indicated by PGP 9.5; C: Immunofluorescence showing the effect of HuD plasmid transfection in counteracting HuD autoantibody-induced cleaved caspase 3 expression in SH-SY5Y cells. Neurons are indicated by PGP 9.5; D: Statistical analysis of the mean fluorescence intensity per cell using terminal-deoxynucleotidyl transferase mediated nick end labeling immunostaining; E: Statistical analysis of the mean fluorescence intensity per cell using cleaved caspase 3 immunostaining. Data are expressed as mean ± SD. One-way and two-way analysis of variance with Tukey’s multiple comparison test were performed, and n = 3 for each group. Scale bar: 50 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling.

Figure 4 Anti-HuD autoantibody induced neuronal apoptosis in an irritable bowel syndrome rat model. A: Terminal-deoxynucleotidyl transferase mediated nick end labeling of neurons in the ileal plexus of the three groups of rats with neurons indicated by PGP 9.5; B: Cleaved caspase 3 staining of neurons in the ileal plexus of the three groups of rats with enteric neurons indicated by PGP 9.5; C: Percentage of apoptotic neurons by terminal-deoxynucleotidyl transferase mediated nick end labeling; D: Percentage of apoptotic neurons by cleaved caspase 3 staining. The yellow arrows indicate apoptotic enteric neurons. Scale bar: 20 μm. Data are expressed as mean ± SD. C and D were analyzed by two-way analysis of variance with Tukey’s multiple comparison test, and n = 3 for each group. ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. DAPI: 4’,6-diamidino-2-phenylindole; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling.

Figure 5 Effect of intervention in primary enteric neurons and the irritable bowel syndrome rate model. A: Effect of neutralizing antibodies by recombinant HuD protein on enteric neurons; B: Effect of protein kinase C agonist, Bryostatin1, 5-hydroxytryptamine agonist and antagonist, and immunoglobulin on enteric neurons; C: PGP 9.5/terminal-deoxynucleotidyl transferase mediated nick end labeling/4’,6-diamidino-2-phenylindole staining of neurons in ileal paraffin sections from the protein kinase C agonist group, the intravenous immunoglobulin group, and the anti-HuD autoantibody group; n = 3 for each group. Scale bar: 20 μm. Data are expressed as mean ± SD. One-way and two-way analysis of variance with Tukey’s multiple comparison test were performed. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IVIG: Immunoglobulin; PKC: Protein kinase C; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 6 mRNA expression of the downstream targets of HuD in rat ileum and colon. A: Special AT-rich sequence-binding protein 1; B: Nerve growth factor; C: Brain-derived neurotrophic factor; D: Neurotrophic factor-3; E: Growth-associated protein-43; F: Neuroserpin. Data are expressed as mean ± SD. One-way analysis of variance with Tukey’s multiple comparison test was performed and n = 3. ^aP < 0.05, ^bP < 0.01, and ^dP < 0.0001. SATB1: Special AT-rich sequence-binding protein 1; NGF: Nerve growth factor; BDNF: Brain-derived neurotrophic factor; NT-3: Neurotrophic factor-3; GAP-43: Growth-associated protein-43.

Figure 7 Anti-HuD autoantibody induced neuronal apoptosis through the special AT-rich sequence-binding protein 1-phosphatidylinositol 3-kinase-protein kinase B pathway. A: Western blot showing the elevated expression of HuD and special AT-rich sequence-binding protein 1 (SATB1) in the Bryostatin1 group (n = 3); B: Confocal immunofluorescence of HuD and SATB1 in rat ileal neurons (n = 3); C: Western blot showing HuD and SATB1 upregulation attenuated apoptosis through the phosphatidylinositol 3-kinase-protein kinase B pathway; D: Confocal immunofluorescence of HuD and SATB1 in SH-SY5Y neurons; E: Western blot showing that SATB1 was necessary for HuD autoantibody-induced apoptosis within the phosphatidylinositol 3-kinase-protein kinase B pathway. Data are expressed as mean ± SD. Scale bar: 30 μm. Two-way analysis of variance with Tukey’s multiple comparison test was performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. DAPI: 4’,6-diamidino-2-phenylindole; SATB1: Special AT-rich sequence-binding protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; p-AKT: Phosphorylated protein kinase B.