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Detrusor myocyte autophagy protects the bladder function via inhibiting the inflammation in cyclophosphamide-induced cystitis in rats.

Zhao J, Song Q, Wang L, Dong X, Yang X, Bai X, Song B, Damaser M, Li L - PLoS ONE (2015)

Bottom Line: The expressions of microtubule-associated protein 1 light chain 3 (LC3), p-p70s6k (the phosphorylated form of ribosomal protein S6), SOD2 (superoxide dismutase 2) in the bladder muscular layer were measured using western blot.Inflammation and oxidative stress were significantly decreased and the bladder histology and micturition function were significantly improved with rapamycin (RAPA, autophagy agonist) pre-treatment.The autophagy agonist RAPA significantly decreased the inflammation and protected the bladder function, which might be considered as a potential treatment for interstitial cystitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Autophagy, a highly conserved homeostatic cellular process that removes and recycles damaged proteins and organelles in response to cellular stress, is believed to play a crucial role in the immune response and inflammation. The role of autophagy in bladder cystitis, however, has not well been clarified. Here we investigate the role of detrusor myocytes autophagy (DMA) in cyclophosphamide-induced cystitis animal model. 164 female Sprague-Dawley rats were randomized into three experimental groups and compared to three control groups, respectively. The expressions of microtubule-associated protein 1 light chain 3 (LC3), p-p70s6k (the phosphorylated form of ribosomal protein S6), SOD2 (superoxide dismutase 2) in the bladder muscular layer were measured using western blot. The co-location of LC3, alpha-smooth muscle actin (α-SMA), and autophagic vacuoles were investigated with double-labeled immunofluorescence and transmission electron microscopy (TEM). The expression of lL-1β, IL-6, IL-8, malondialdehyde (MDA), and glutathione (GSH) in the detrusor layer were analyzed using ELISA. The bladder inflammation and the number of mast cells in the muscular layer were analyzed by histology. The bladder function was evaluated using cystometry. In cyclophosphamide-induced cystitis, autophagy was detected in detrusor myocytes by increased LC3, p-p70s6k expression, and autophagosomes. However, the presence of enhanced inflammation and oxidative stress in the cyclophosphamide-treated group suggest autophagy of detrusor myocytes may not be sufficiently activated. Inflammation and oxidative stress were significantly decreased and the bladder histology and micturition function were significantly improved with rapamycin (RAPA, autophagy agonist) pre-treatment. In contrast, inflammation and oxidative stress were dramatically increased and the bladder histology and function were negatively affected with chloroquine (CQ, autophagy blocker) pre-treated. These findings preferentially provide evidence of the association between DMA and cyclophosphamide-induced cystitis in rats. The autophagy agonist RAPA significantly decreased the inflammation and protected the bladder function, which might be considered as a potential treatment for interstitial cystitis.

No MeSH data available.


Related in: MedlinePlus

Evaluation of IL-1β, IL-6 and IL-8 changes in the bladder muscular layer in CYP-treated rats using ELISA kits.(A) IL-1β expression, (B) IL-6 expression and (C) IL-8 expression, n = 5. Data are expressed as the mean ± SD, a indicates a significant difference from normal at each time point (P <0.05). b indicates a significant difference compared to the CYP group value (P <0.05).
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pone.0122597.g004: Evaluation of IL-1β, IL-6 and IL-8 changes in the bladder muscular layer in CYP-treated rats using ELISA kits.(A) IL-1β expression, (B) IL-6 expression and (C) IL-8 expression, n = 5. Data are expressed as the mean ± SD, a indicates a significant difference from normal at each time point (P <0.05). b indicates a significant difference compared to the CYP group value (P <0.05).

Mentions: Expression of IL-1β, IL-6, IL-8, and MDA in the bladder muscular layer was significantly increased in the CYP, CYP+RAPA, and CYP+CQ groups at each time point compared to sham CYP, RAPA, and CQ groups, respectively (Figs 4 and 5). Expression of these substances was significantly decreased in the CYP +RAPA group and significantly increased in the CYP+CQ group compared to the CYP group, however. The antioxidant GSH and antioxidant enzyme SOD2 present in the muscular layer was significantly reduced in the CYP group at the 4 h time point but showed an increasing trend at 48 h and 72 h. GSH and SOD2 levels in the CYP +RAPA group were significantly enhanced compared to the CYP group at all time points, and were significantly increased compared to the RAPA group at 48 h and 72 h. In addition, compared to the CQ group, GSH levels in the CYP+CQ group was significantly decreased at all time points. SOD2 was significantly decreased at 4 h and 72 h (Fig 5). Evaluation of both GSH and SOD2 in CYP+CQ group indicated significant differences compared to the CYP group at 48 h and 72 h.


Detrusor myocyte autophagy protects the bladder function via inhibiting the inflammation in cyclophosphamide-induced cystitis in rats.

Zhao J, Song Q, Wang L, Dong X, Yang X, Bai X, Song B, Damaser M, Li L - PLoS ONE (2015)

Evaluation of IL-1β, IL-6 and IL-8 changes in the bladder muscular layer in CYP-treated rats using ELISA kits.(A) IL-1β expression, (B) IL-6 expression and (C) IL-8 expression, n = 5. Data are expressed as the mean ± SD, a indicates a significant difference from normal at each time point (P <0.05). b indicates a significant difference compared to the CYP group value (P <0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4382282&req=5

pone.0122597.g004: Evaluation of IL-1β, IL-6 and IL-8 changes in the bladder muscular layer in CYP-treated rats using ELISA kits.(A) IL-1β expression, (B) IL-6 expression and (C) IL-8 expression, n = 5. Data are expressed as the mean ± SD, a indicates a significant difference from normal at each time point (P <0.05). b indicates a significant difference compared to the CYP group value (P <0.05).
Mentions: Expression of IL-1β, IL-6, IL-8, and MDA in the bladder muscular layer was significantly increased in the CYP, CYP+RAPA, and CYP+CQ groups at each time point compared to sham CYP, RAPA, and CQ groups, respectively (Figs 4 and 5). Expression of these substances was significantly decreased in the CYP +RAPA group and significantly increased in the CYP+CQ group compared to the CYP group, however. The antioxidant GSH and antioxidant enzyme SOD2 present in the muscular layer was significantly reduced in the CYP group at the 4 h time point but showed an increasing trend at 48 h and 72 h. GSH and SOD2 levels in the CYP +RAPA group were significantly enhanced compared to the CYP group at all time points, and were significantly increased compared to the RAPA group at 48 h and 72 h. In addition, compared to the CQ group, GSH levels in the CYP+CQ group was significantly decreased at all time points. SOD2 was significantly decreased at 4 h and 72 h (Fig 5). Evaluation of both GSH and SOD2 in CYP+CQ group indicated significant differences compared to the CYP group at 48 h and 72 h.

Bottom Line: The expressions of microtubule-associated protein 1 light chain 3 (LC3), p-p70s6k (the phosphorylated form of ribosomal protein S6), SOD2 (superoxide dismutase 2) in the bladder muscular layer were measured using western blot.Inflammation and oxidative stress were significantly decreased and the bladder histology and micturition function were significantly improved with rapamycin (RAPA, autophagy agonist) pre-treatment.The autophagy agonist RAPA significantly decreased the inflammation and protected the bladder function, which might be considered as a potential treatment for interstitial cystitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Autophagy, a highly conserved homeostatic cellular process that removes and recycles damaged proteins and organelles in response to cellular stress, is believed to play a crucial role in the immune response and inflammation. The role of autophagy in bladder cystitis, however, has not well been clarified. Here we investigate the role of detrusor myocytes autophagy (DMA) in cyclophosphamide-induced cystitis animal model. 164 female Sprague-Dawley rats were randomized into three experimental groups and compared to three control groups, respectively. The expressions of microtubule-associated protein 1 light chain 3 (LC3), p-p70s6k (the phosphorylated form of ribosomal protein S6), SOD2 (superoxide dismutase 2) in the bladder muscular layer were measured using western blot. The co-location of LC3, alpha-smooth muscle actin (α-SMA), and autophagic vacuoles were investigated with double-labeled immunofluorescence and transmission electron microscopy (TEM). The expression of lL-1β, IL-6, IL-8, malondialdehyde (MDA), and glutathione (GSH) in the detrusor layer were analyzed using ELISA. The bladder inflammation and the number of mast cells in the muscular layer were analyzed by histology. The bladder function was evaluated using cystometry. In cyclophosphamide-induced cystitis, autophagy was detected in detrusor myocytes by increased LC3, p-p70s6k expression, and autophagosomes. However, the presence of enhanced inflammation and oxidative stress in the cyclophosphamide-treated group suggest autophagy of detrusor myocytes may not be sufficiently activated. Inflammation and oxidative stress were significantly decreased and the bladder histology and micturition function were significantly improved with rapamycin (RAPA, autophagy agonist) pre-treatment. In contrast, inflammation and oxidative stress were dramatically increased and the bladder histology and function were negatively affected with chloroquine (CQ, autophagy blocker) pre-treated. These findings preferentially provide evidence of the association between DMA and cyclophosphamide-induced cystitis in rats. The autophagy agonist RAPA significantly decreased the inflammation and protected the bladder function, which might be considered as a potential treatment for interstitial cystitis.

No MeSH data available.


Related in: MedlinePlus