Aldh2 gene reduces oxidative stress in the bladder by regulating the NF-κB pathway in a mouse model of ketamine-induced cystitis

Abstract

Aldehyde dehydrogenase 2 (aldh2) serves an important role in the development of organ injury. Therefore, the present study investigated the effects of aldh2 on the oxidative stress response in a mouse model of ketamine-induced cystitis (KIC). A total of 60 8-week-old male Institute of Cancer Research wild-type (WT) mice and 45 aldh2 knock-out (KO) mice were randomized to receive low-dose ketamine (30 mg/kg), high-dose ketamine (60 mg/kg) or normal saline (controls). At 4, 8 and 12 weeks post-injection, bladder tissues were harvested and used to investigate the protective mechanisms of aldh2 on bladder function. The results demonstrated that aldh2 KO mice exhibited significant weight loss following chronic ketamine injection compared with that in WT mice. Furthermore, ketamine treatment increased the urination rate (P<0.05), pathological score (P<0.05), levels of the oxidative stress product malondialdehyde (P<0.05) in addition to reducing the expression of the anti-oxidative stress enzyme superoxide dismutase (P<0.05) and glutathione-SH (P<0.05). Oxidative stress in aldh2 KO mice was also found to significantly enhance the expression of proteins associated with the NF-κB signaling pathway, which promoted the expression of inducible nitric oxide synthase (P<0.05) and cyclooxygenase-2 (P<0.05) further. Finally, aldh2 KO mice demonstrated higher severity of fibrosis in the submucosal and muscular layers of the bladder. In conclusion, the present study suggests that aldh2 serves a protective role in preventing inflammation and fibrosis in KIC.

Keywords: aldehyde dehydrogenase 2; cyclooxygenase-2; inducible nitric oxide synthase; ketamine-induced cystitis; oxidative stress.

Figure 1

Ketamine treatment increases aldh2 mRNA expression in WT mice and increases urination frequency and reduces body weight in KO mice. (A) Reverse transcription-quantitative PCR was performed to measure the mRNA expression of aldh2 in WT mice at 4 and 8 weeks following saline and ketamine treatment. (B) Changes in urination frequency of KO and WT mice during 4-, 8- and 12-week ketamine treatments. (C) Weekly weight changes of KO and WT mice (each week, group WT vs. group KO). Data were presented as the mean ± standard error of the mean from ≥3 experimental repeats. ^*P<0.05 and ^**P<0.01. Aldh2, aldehyde dehydrogenase 2; WT, wild-type; KO, knock-out; WNS, wild-type normal saline control group; WLK, wild-type low-dose ketamine group; WHK, wild-type high-dose ketamine group; KNS, knock-out normal saline control group; KLK, knock-out low-dose ketamine group; KHK, knock-out high-dose ketamine group; NS, normal saline; LK, low-dose ketamine; HK, high-dose ketamine; w, week.

Figure 2

Ketamine increases the level of oxidative stress in aldh2 KO mice and aggravates pathological damage. (A) Effects of ketamine on parameters of oxidative stress in WT and KO mice at 4, 8 and 12 weeks as detected by ELISA. Negative control mice were treated with NS. ^*P<0.05 and ^**P<0.01. KO, knock-out; NS, normal saline; KHK, knock-out high-dose ketamine group; SOD, superoxide dismutase; GSH, glutathione-sulfhydryl; MDA, malondialdehyde; WHK, wild-type high-dose ketamine group; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; WT, wild-type; KNS, knock-out normal saline control group; WNS, wild-type normal saline control group; KLK, knock-out low-dose ketamine group; WLK, wild-type low-dose ketamine group; HK, high-dose ketamine; W, week; -, knock-out; +, wild-type. Ketamine increases the level of oxidative stress in aldh2 KO mice and aggravates pathological damage. (B) Representative hematoxylin and eosin staining images of bladder tissues from KO and WT mice in week 12. Magnification, x100 for the upper images; x400 for the lower images. (C) Representative immunohistochemical staining images of COX-2 and iNOS proteins in the bladder tissues of KO and WT mice in weeks 4 and 12. The cytoplasm and cell membranes exhibiting brown-yellow colors were considered as positive expression of the target protein, which were mainly confined to the bladder epithelium Magnification, x200. (D) Representative Masson trichrome staining images of bladder tissues of KO and WT mice in week 12. Collagen fibers stained green, muscle fibers stained red, and nucleus stained blue-brown. Magnification, x200. Data are presented as the mean ± standard error of the mean from ≥3 experimental repeats. ^*P<0.05 and ^**P<0.01. KO, knock-out; NS, normal saline; KHK, knock-out high-dose ketamine group; SOD, superoxide dismutase; GSH, glutathione-sulfhydryl; MDA, malondialdehyde; WHK, wild-type high-dose ketamine group; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; WT, wild-type; KNS, knock-out normal saline control group; WNS, wild-type normal saline control group; KLK, knock-out low-dose ketamine group; WLK, wild-type low-dose ketamine group; HK, high-dose ketamine; W, week; -, knock-out; +, wild-type.

Figure 3

Ketamine treatment induces inflammation and fibrosis in KO mice. (A) Effects of ketamine on inflammation protein COX-2, iNOS and NF-κB levels in mice. ^*P<0.05 and ^**P<0.01. Data are presented as the mean ± standard error of the mean from ≥3 experimental repeats. KO, knock-out; KHK, KO high-dose ketamine group; WHK, wild-type high-dose ketamine group; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; WT, wild-type; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor β; KNS, knock-out normal saline control group; WNS, wild-type normal saline control group; KLK, knock-out low-dose ketamine group; WLK, wild-type low-dose ketamine group; W, weeks; -, knock-out; +, wild-type; HK, high-dose ketamine. Ketamine treatment induces inflammation and fibrosis in KO mice. (B) mRNA expression levels of inflammatory proteins COX-2, iNOS and NF-κB in the mouse bladder tissues were detected by reverse transcription-quantitative PCR. (C) Western blotting and (D) reverse transcription-quantitative PCR were performed to measure the expression of α-SMA, TGF-β and fibronectin in mouse bladder tissues at week 12. ^*P<0.05 and ^**P<0.01. Data are presented as the mean ± standard error of the mean from ≥3 experimental repeats. KO, knock-out; KHK, KO high-dose ketamine group; WHK, wild-type high-dose ketamine group; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; WT, wild-type; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor β; KNS, knock-out normal saline control group; WNS, wild-type normal saline control group; KLK, knock-out low-dose ketamine group; WLK, wild-type low-dose ketamine group; W, weeks; -, knock-out; +, wild-type; HK, high-dose ketamine.