Antibodies Monoclonal mouse antibodies against TNF-a (sc-80383), b-actin (sc-47778), NF-kB (sc-8008), and pNF-kB (sc-135768) and polyclonal rabbit antibodies against IL-1b, (sc-7884), NALP1 (sc-66846), NALP3 (sc-66992), caspase-1 (sc-515), BAX (sc-493), Caspase-3 (sc-7148) cleaved caspase-3, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-20357) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA)

Antibodies Monoclonal mouse antibodies against TNF-a (sc-80383), b-actin (sc-47778), NF-kB (sc-8008), and pNF-kB (sc-135768) and polyclonal rabbit antibodies against IL-1b, (sc-7884), NALP1 (sc-66846), NALP3 (sc-66992), caspase-1 (sc-515), BAX (sc-493), Caspase-3 (sc-7148) cleaved caspase-3, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-20357) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). the carrageenan-induced hyperalgesia and reduced the elevated levels of proteins including TNF-a and IL-1b in the rats. Apoptosis markers, B-cell lymphoma 2-associated X protein (Bax) and caspase-3, were elevated in ICTA-treated Chronic pelvic pain syndrome (CPPS) rats. These results suggest that ICTA provides protection against local carrageenan-induced enhanced pain sensitivity, and that the neutralization of proinflammatory cytokines may result in inflammatory cell apoptosis. = 3). (C) In vitro detection of the tumor necrosis factor-alpha (TNF-a) signals by TNF-a-targeting aptamer (AptTNF-a) or a random sequence pool in a plate fixed with human TNF-a (= 3). (D) Dose-dependent binding of AptIL-1b to IL-1b demonstrated a standard curve in enzyme-linked immunosorbent assay (ELISA) by replacing primary antibody with AptIL-1b. (E) The proliferation of the Jurkat cell line detected with Cell Counting Kit-8 (CCK8, Sigma-Aldrich, Product No. 96992) at 48 h after IL-1b with/without aptamer administration (= 3). (F) The proliferation of murine urothelial cell line detected with CCK8 at 48 h after the administration of TNF-a with/without aptamer (= 3). The data are presented as mean standard error of the mean and were analyzed by Students t-test. Asterisks denote statistically significant differences. * 0.05 represents a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 represents a significant difference for the CPPS + ICTA group compared with the CPPS group ( 0.05). 2.2. ICTA Attenuated Local Carrageenan-Induced Mechanical Allodynia and Tactile Hyperalgesia All male rats received intraprostatic injection with control vehicle, carrageenan (CPPS group), carrageenan with ICTA (CPPS + ICTA group), or ICTA alone. Mechanical allodynia was assessed using the von Frey filament test at both the scrotal wall and the tail base. Consistent with our previous study, local carrageenan intraprostatic injection to SD rats resulted in a lower pain threshold at the scrotal wall of SD rats compared with that of the control group ( 0.05) (Figure 2A). ICTA treatment significantly reduced the carrageenan-induced mechanical allodynia in SD rats ( 0.05) (Figure 2A). The pain threshold at the tail base was not reduced significantly (Figure 2B). Open in a separate window Figure 2 Inflammatory cytokine-targeting aptamers (ICTA) attenuated local carrageenan-induced hypersensitivity of scrotal wall (A) and tail base (B) allodynia in the von Frey filament test of Sprague Dawley (SD) rats. (A) The CPPS + ICTA group compared with the chronic pelvic pain syndrome (CPPS) group (= 6). Local carrageenan treatment resulted in reduction of mean responsive intensity in tail reaction from tactile stimulation in the SD rats. Treatment with ICTA significantly reduced carrageenan-induced pain hypersensitivity in the SD rats. The results are expressed as the mean standard error of the mean (SEM) of six animals in each group and were analyzed by one-way analysis of variance (ANOVA). * 0.05 represents a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 represents a significant difference for the CPPS + ICTA group compared with the CPPS group (= 6). 2.3. ICTA Modified Local Carrageenan-Induced Glandular Hyperplasia and Inflammatory Responses in the Prostate Local carrageenan treatment stimulated hyperplasia of glandular epithelium in the prostate as indicated by Haemotoxylin& Eosin( 0.05) (Figure 3O). IL-1b expression was lowered in CPPS + ICTA group compared to CPPS group, but not significantly (Figure 3J). Open in a separate window Figure 3 Inflammatory cytokine-targeting aptamers (ICTA) affected carrageenan-induced mononuclear Isosilybin A cell infiltration, as assessed by interleukin-1 beta (IL-1b), tumor necrosis factor-alpha TNF-a, and Caspase-1 staining, in the prostate glandular epithelium of the Sprague Dawley rats (SD rats). The scheme used an Haemotoxylin& Eosin( 0. 05 represents significance for the CPPS group or CPPS + ICTA group compared with the control group. ** 0.05 represents significance for the CPPS + ICTA group compared with the CPPS group (= 6). Local exposure to carrageenan resulted in inflammasome activation in rat prostate, as evidenced by the increases in NALP1+ cells (Figure 4B) and NALP3+ cells (Figure 4G). Seven days after carrageenan injection, NALP1 and NALP3 protein expression in the prostate of carrageenan-exposed rats (CPPS) dramatically increased compared with that in the saline-injected control rats ( 0.05) (Figure 4A,F). ICTA attenuated the carrageenan-induced increase in the concentration levels of NALP1 in the prostate of rats ( 0.05) (Figure 4E). However, NALP3 expression in the CPPS + ICTA group prostate was significantly increased compared with that in the control and CPPS groups ( 0.05) (Figure.Local carrageenan administration resulted in a reduction of the tactile threshold. ICTA significantly attenuated the carrageenan-induced hyperalgesia and reduced the elevated levels of proteins including TNF-a and IL-1b in the rats. Apoptosis markers, B-cell lymphoma 2-associated X protein (Bax) and caspase-3, were elevated in ICTA-treated Chronic pelvic pain syndrome (CPPS) rats. These results suggest that ICTA provides safety against local carrageenan-induced enhanced pain sensitivity, and that the neutralization of proinflammatory cytokines may result in inflammatory cell apoptosis. = 3). (C) In vitro detection of the tumor necrosis factor-alpha (TNF-a) signals by TNF-a-targeting aptamer (AptTNF-a) or a random sequence pool inside a plate fixed with human being TNF-a (= 3). (D) Dose-dependent binding of AptIL-1b to IL-1b shown a standard curve in enzyme-linked immunosorbent assay (ELISA) by replacing main antibody with AptIL-1b. (E) The proliferation of the Jurkat cell collection recognized with Cell Counting Kit-8 (CCK8, Sigma-Aldrich, Product No. 96992) at 48 h after IL-1b with/without aptamer administration (= 3). (F) The proliferation of murine urothelial cell collection recognized with CCK8 at 48 h after the administration of TNF-a with/without aptamer (= 3). The data are offered as mean standard error of the mean and were analyzed by College students t-test. Asterisks denote statistically significant variations. * 0.05 signifies a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 signifies a significant difference for the CPPS + ICTA group compared with the CPPS group ( 0.05). 2.2. ICTA Attenuated Local Carrageenan-Induced Mechanical Allodynia and Tactile Hyperalgesia All male rats received intraprostatic injection with control vehicle, carrageenan (CPPS group), carrageenan with ICTA (CPPS + ICTA group), or ICTA only. Mechanical allodynia was assessed using the von Frey filament test at both the scrotal wall and the tail foundation. Consistent with our earlier study, local carrageenan intraprostatic injection to SD rats resulted in a lower pain threshold in the scrotal wall of SD rats compared with that of the control group ( 0.05) (Figure 2A). ICTA treatment significantly reduced the carrageenan-induced mechanical allodynia in SD rats ( 0.05) (Figure 2A). The pain threshold in the tail foundation was not reduced significantly (Number 2B). Open in a separate window Number 2 Inflammatory cytokine-targeting aptamers (ICTA) attenuated local carrageenan-induced hypersensitivity of scrotal wall (A) and tail foundation (B) allodynia in the von Frey filament test of Sprague Dawley (SD) rats. (A) The CPPS + ICTA group compared with the chronic pelvic pain syndrome (CPPS) group (= 6). Local carrageenan treatment resulted in reduction of mean responsive intensity in tail reaction from tactile activation in the SD rats. Treatment with ICTA significantly reduced carrageenan-induced pain hypersensitivity in the SD rats. The results are indicated as the mean standard error of the mean (SEM) of six animals in each group and were analyzed by one-way analysis of variance (ANOVA). * 0.05 signifies a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 signifies a significant difference for the CPPS + ICTA group compared with the CPPS group (= 6). 2.3. ICTA Modified Local Carrageenan-Induced Glandular Hyperplasia and Inflammatory Reactions in the Prostate Local carrageenan treatment stimulated hyperplasia of glandular epithelium in the prostate as indicated by Haemotoxylin& Eosin( 0.05) (Figure 3O). IL-1b manifestation was lowered in CPPS + ICTA group compared to CPPS group, but not significantly (Number 3J). Open in a separate window Number 3 Inflammatory cytokine-targeting aptamers (ICTA) affected carrageenan-induced mononuclear cell infiltration, as assessed by interleukin-1 beta (IL-1b), tumor necrosis factor-alpha TNF-a, and Caspase-1 staining, in the prostate glandular epithelium of the Sprague Dawley rats (SD Isosilybin A rats). The plan used an Haemotoxylin& Eosin( 0.05 signifies significance for the CPPS group or.These histological and behavioral changes can be revised by ICTA (IL-1b- and TNF-a-targeting aptamers), and the preventive effects may be associated with its ability to neutralize carrageenan-induced pro-inflammatory cytokines and apoptosis activation in interstitial mononuclear cells. The levels of mononuclear cell infiltration, pro-inflammatory cytokine interleukin-1 beta (b), caspase-1 (casp-1), and Nucleotide-binding oligomerization website, Leucine rich Repeat and Pyrin website comprising proteins 1 and 3 (NALP1 and NALP3) in the prostate of rats were increased seven days after carrageenan injection. Treatment with ICTA significantly attenuated the carrageenan-induced hyperalgesia and reduced the elevated levels of proteins including TNF-a and IL-1b in the rats. Apoptosis markers, B-cell lymphoma 2-connected X protein (Bax) and caspase-3, were elevated in ICTA-treated Chronic pelvic pain syndrome (CPPS) rats. These results suggest that ICTA provides safety against local carrageenan-induced enhanced pain sensitivity, and that the neutralization of proinflammatory cytokines may result in inflammatory cell apoptosis. = 3). (C) In vitro detection of the tumor necrosis factor-alpha (TNF-a) signals by TNF-a-targeting aptamer (AptTNF-a) or a random sequence pool inside a plate fixed with human being TNF-a (= 3). (D) Dose-dependent binding of AptIL-1b to IL-1b shown a standard curve in enzyme-linked immunosorbent assay (ELISA) by replacing main antibody with AptIL-1b. (E) The proliferation of the Jurkat cell collection recognized with Cell Counting Kit-8 (CCK8, Sigma-Aldrich, Product No. 96992) at 48 h after IL-1b with/without aptamer administration (= 3). (F) The proliferation of murine urothelial cell collection recognized with CCK8 at 48 h after the administration of TNF-a with/without aptamer (= 3). The data are offered as mean standard error of Isosilybin A the mean and were analyzed by College students t-test. Asterisks denote statistically significant variations. * 0.05 signifies a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 represents a significant difference for the CPPS + ICTA group compared with the CPPS group ( 0.05). 2.2. ICTA Attenuated Local Carrageenan-Induced Mechanical Allodynia and Tactile Hyperalgesia All male rats received intraprostatic injection with control vehicle, carrageenan (CPPS group), carrageenan with ICTA (CPPS + ICTA group), or ICTA alone. Mechanical allodynia was assessed using the von Frey filament test at both the scrotal wall and the tail base. Consistent with our previous study, local carrageenan intraprostatic injection to SD rats resulted in a lower pain threshold at the scrotal wall of SD rats compared with that of the control group ( 0.05) (Figure 2A). ICTA treatment significantly reduced the carrageenan-induced mechanical allodynia in SD rats ( 0.05) (Figure 2A). The pain threshold at the tail base was not reduced significantly (Physique 2B). Open in a separate window Physique 2 Inflammatory cytokine-targeting aptamers (ICTA) attenuated local carrageenan-induced hypersensitivity of scrotal wall (A) and tail base (B) allodynia in the von Frey filament test of Sprague Dawley (SD) rats. (A) The CPPS + ICTA group compared with the chronic pelvic pain syndrome (CPPS) group (= 6). Local carrageenan treatment resulted in reduction of Isosilybin A mean responsive intensity in tail reaction from tactile activation in the SD rats. Treatment with ICTA significantly reduced carrageenan-induced pain hypersensitivity in the SD rats. The results are expressed as the mean standard error of the mean (SEM) of six animals in each group and were analyzed by one-way analysis of variance (ANOVA). * 0.05 represents a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 represents a significant difference for the CPPS + ICTA group compared with the CPPS group (= 6). 2.3. ICTA Modified Local Carrageenan-Induced Glandular Hyperplasia and Inflammatory Responses in the Prostate Local carrageenan treatment stimulated hyperplasia of glandular epithelium in the prostate as indicated by Haemotoxylin& Eosin( 0.05) (Figure 3O). IL-1b expression was lowered in CPPS + ICTA group compared to CPPS group, but not significantly (Physique 3J). Open in a separate window Physique 3 Inflammatory cytokine-targeting aptamers (ICTA) affected carrageenan-induced mononuclear cell infiltration, as assessed by interleukin-1 beta (IL-1b), tumor necrosis factor-alpha TNF-a, and Caspase-1 staining, in the prostate glandular epithelium of the Sprague Dawley rats (SD rats). The plan used an Haemotoxylin& Eosin( 0.05 represents significance for the CPPS group or CPPS + ICTA group compared with the control group. ** 0.05.(ACD) Immunohistochemical staining results show that ICTA prominently promoted pro-caspase-3 expression and cleavage in CPPS prostate (C) compared with those in the CPPS only (B) and control groups (A). rich Repeat and Pyrin domain name made up of proteins 1 and 3 (NALP1 and NALP3) in the prostate Rabbit Polyclonal to MED14 of rats were increased seven days after carrageenan injection. Treatment with ICTA significantly attenuated the carrageenan-induced hyperalgesia and reduced the elevated levels of proteins including TNF-a and IL-1b in the rats. Apoptosis markers, B-cell lymphoma 2-associated X protein (Bax) and caspase-3, were elevated in ICTA-treated Chronic pelvic pain syndrome (CPPS) rats. These results suggest that ICTA provides protection against local carrageenan-induced enhanced pain sensitivity, and that the neutralization of proinflammatory cytokines may result in inflammatory cell apoptosis. = 3). (C) In vitro detection of the tumor necrosis factor-alpha (TNF-a) signals by TNF-a-targeting aptamer (AptTNF-a) or a random sequence pool in a plate fixed with human TNF-a (= 3). (D) Dose-dependent binding of AptIL-1b to IL-1b exhibited a standard curve in enzyme-linked immunosorbent assay (ELISA) by replacing main antibody with AptIL-1b. (E) The proliferation of the Jurkat cell collection detected with Cell Counting Kit-8 (CCK8, Sigma-Aldrich, Product No. 96992) at 48 h after IL-1b with/without aptamer administration (= 3). (F) The proliferation of murine urothelial cell collection detected with CCK8 at 48 h after the administration of TNF-a with/without aptamer (= 3). The data are offered as mean standard error of the mean and were analyzed by Students t-test. Asterisks denote statistically significant differences. * 0.05 represents a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 represents a significant difference for the CPPS + ICTA group compared with the CPPS group ( 0.05). 2.2. ICTA Attenuated Local Carrageenan-Induced Mechanical Allodynia and Tactile Hyperalgesia All male rats received intraprostatic injection with control vehicle, carrageenan (CPPS group), carrageenan with ICTA (CPPS + ICTA group), or ICTA alone. Mechanical allodynia was assessed using the von Frey filament test at both the scrotal wall and the tail base. Consistent with our previous study, local carrageenan intraprostatic injection to SD rats resulted in a lower pain threshold at the scrotal wall of SD rats compared with that of the control group ( 0.05) (Figure 2A). ICTA treatment significantly reduced the carrageenan-induced mechanical allodynia in SD rats ( 0.05) (Figure 2A). The pain threshold at the tail base was not reduced significantly (Physique 2B). Open in a separate window Physique 2 Inflammatory cytokine-targeting aptamers (ICTA) attenuated local carrageenan-induced hypersensitivity of scrotal wall (A) and tail base (B) allodynia in the von Frey filament test of Sprague Dawley (SD) rats. (A) The CPPS + ICTA group compared with the chronic pelvic pain syndrome (CPPS) group (= 6). Local carrageenan treatment resulted in reduction of mean responsive intensity in tail reaction from tactile activation in the SD rats. Treatment with ICTA significantly reduced carrageenan-induced pain hypersensitivity in the SD rats. The results are expressed as the mean standard error of the mean (SEM) of six animals in each group and were analyzed by one-way analysis of variance (ANOVA). * 0.05 represents a significant difference for the CPPS group compared with the sham-operated control group. ** 0.05 represents a significant difference for the CPPS + ICTA group compared with the CPPS group (= 6). 2.3. ICTA Modified Local Carrageenan-Induced Glandular Hyperplasia and Inflammatory Responses in the Prostate Local carrageenan treatment stimulated hyperplasia of glandular epithelium in the prostate as indicated by Haemotoxylin& Eosin( 0.05) (Figure 3O). IL-1b expression was lowered in CPPS + ICTA group compared to CPPS group, but not significantly (Physique 3J). Open.