HO-1 alleviates neuroinflammation and cystitis-related symptoms in cyclophosphamide-induced cystitis via the SIRT1 pathway

doi:10.21203/rs.3.rs-2006593/v1

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HO-1 alleviates neuroinflammation and cystitis-related symptoms in cyclophosphamide-induced cystitis via the SIRT1 pathway

https://doi.org/10.21203/rs.3.rs-2006593/v1

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Background

Our previous study suggested that astrocytes and microglia are activated in the spinal dorsal horn (SDH) of interstitial cystitis/bladder pain syndrome (IC/BPS) rats and induce neuroinflammation by secreting proinflammatory cytokines. Heme oxygenase-1 (HO-1) plays a key role in inhibiting neuroinflammatory processes in the central nervous system and can activate silent information regulator 1 (SIRT1), which has an inhibitory effect on neuroinflammation; however, whether HO-1 alleviates neuroinflammation in IC remains unclear. This study aimed to elucidate the role of HO-1 in rat IC models and confirm whether SIRT1 mediates HO-1 function.

Methods

Rats were administered with cyclophosphamide (CYP) by systemic intraperitoneal injection to develop IC models. Hemin (inducer of HO-1) and Znpp (HO-1 inhibitor) were performed intraperitoneally 1-day prior to each CYP injection. EX-527 was injected intrathecally for 3 consecutive days to selectively inhibit SIRT1. We used the von Frey filament test to measure mechanical withdrawal threshold, and urinary frequency was assessed using urodynamic tests. HO-1, SIRT1, glial fibrillary acidic protein (an astrocyte marker), ionized calcium-binding adapter molecule (a microglia marker), phosphorylated (p)-c-Jun N-terminal kinase (JNK), p-p38, and proinflammatory cytokines [interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α] levels were assessed by western blot, and immunofluorescence was used to identify HO-1 and SIRT1 cellular localization.

Results

We observed downregulated HO-1 expression in the SDH of rats with CYP-induced cystitis, which was accompanied by neuroinflammation, mechanical allodynia, and urinary frequency. Additionally, HO-1 induction after hemin treatment suppressed glial cell activation and attenuated IL-1β, IL-6, and TNF-α expression by inhibiting activation of the JNK/p38 pathway, ultimately improving IC-related symptoms. Moreover, Znpp administration exacerbated inflammatory responses and pain sensitivity by inhibiting HO-1 activity. Furthermore, HO-1 positively regulated SIRT1 activation and alleviated IC-related symptoms, whereas the therapeutic effect of HO-1 upregulation was significantly impaired by SIRT1 inhibition.

Conclusion

HO-1 attenuated neuroinflammation, mechanical allodynia, and urinary frequency caused by glial activation in rats with CYP-induced cystitis by activating SIRT1 to inhibit JNK/p38 signaling.

interstitial cystitis/bladder pain syndrome

heme oxygenase-1

silent information regulator 1

neuroinflammation

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory condition of the bladder that is accompanied by frequent urination, pelvic pain, and nocturia [1]. In severe cases, patients can develop comorbid cognitive and psychiatric disorders [2, 3]. An estimated 2.7–6.5% of American females suffer from IC symptoms [4, 5], which significantly reduces their quality of life and results in significant economic burdens [6]. Unfortunately, the lack of understanding of IC pathogenesis and etiology has resulted in largely ineffective therapies. We previously suggested that neuroglial cells (microglia and astrocytes) may respond to exogenous stimuli by producing proinflammatory factors, ultimately resulting in neuroinflammation of the spinal dorsal horn (SDH), which might contribute to mechanical allodynia and facilitate IC development [7, 8]. Therefore, suppression of neuroinflammation in SDH might represent a beneficial strategy for IC treatment.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in heme catabolism and exerts anti-apoptotic, anti-inflammatory, and cytoprotective activities [9]. HO-1 is an inducible enzyme [10], the activity of which can be upregulated in heme catabolism to convert heme to ferrous ion, carbon monoxide (CO), and biliverdin, which is quickly transformed into the powerful antioxidant bilirubin by biliverdin reductase [11–13]. A previous study suggests that HO-1 plays an important role in multiple pathological models involved in dampening inflammation and analgesia [14]. Additionally, another study identified HO-1-mediated anti-inflammatory effects in humans with HO-1 deficiency caused by genetic mutations [15]. Moreover, evidence linked imbalances in HO-1 activity to neuroinflammatory diseases [16], and HO-1 inhibits neuroinflammatory processes and provides neuroprotection in the central nervous system [17, 18]. However, the role of HO-1 in restraining neuroinflammatory responses and alleviating pelvic pain in IC remains uncertain.

Sirtuins, a class of regulatory molecules involved in inflammation, are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases. When a sirtuin enzyme binds an acetylated substrate, the interconnecting glycosidic bond between NAD+-linking nicotinamide and ADP-ribose is cleaved and combines with the acetyl moiety of the substrate to form acetyl-ADP-ribose, leaving a deacetylated protein [19]. Silent information regulator 1 (SIRT1) is the first sirtuin that has the largest terminal regions extensions. SIRT1 is generally considered a nuclear protein [20]; however, studies show that numerous SIRT1 molecules can also shuttle between the nucleus and cytoplasm [21]. SIRT1 is widespread in the central nervous system (CNS) [22, 23] and highly expressed in the spinal cord [24]. Additionally, the suppressive effect of SIRT1 on neuroinflammation has been reported in multiple disease models, including those of cerebral ischemia [25], traumatic brain injury [26], spinal cord injury [27], Alzheimer’s disease [28], and Parkinson’s disease [29]. Therefore, SIRT1 plays a critical role in inhibiting neuroinflammation in the CNS.

Numerous studies report the involvement of HO-1 and SIRT1 in inhibiting inflammation [30, 31]. Gao et al. [32] showed that high glucose (HG) can facilitate inflammatory responses in endothelial progenitor cells (EPCs), during which the expression of molecules in the HO-1/SIRT1 signaling pathway was reduced; however, HO-1 upregulation inhibited inflammation in HG-induced EPCs by activating the SIRT1 pathway. Additionally, a previous study suggested that fenofibrate, a peroxisome proliferator-activated receptor α-specific agonist, upregulated HO-1 expression in glioblastoma cells to activate SIRT1, resulting in SIRT1 accumulation in the nucleus[33]. However, whether HO-1 attenuates neuroinflammation in the SDH of IC rats through the SIRT1 signaling pathway remains unknown.

To investigate the underlying regulatory mechanism of and effective therapeutic strategies for IC, we evaluated the suppressive effect of HO-1 on neuroinflammation in a rat model of cyclophosphamide (CYP)-induced cystitis. Furthermore, we determined whether HO-1 alleviates neuroinflammation, mechanical allodynia, and urinary frequency in CYP-induced cystitis by activating the SIRT1 signaling pathway.

Animals

Female Sprague–Dawley rats (200–220 g) were provided by the Institute of Experimental Animals at Sun Yat-sen University (Guangzhou, China). All rats were housed in a constant environment (independent ventilation, controlled temperature and humidity, 12-h light/dark cycle) and allowed free access to food and water. All experiments conformed with the Guide for the Care and Use of Laboratory Animals at the Institute of Laboratory Animal Research of Sun Yat-sen University.

Reagents

We used CYP (Sigma-Aldrich, St. Louis, MO, USA) to establish the cystitis model, as previously described [34, 35]. Briefly, rats received intraperitoneal (i.p.) injections of CYP (50 mg/kg on days 1, 4, and 7. Injections (i.p.) of hemin (HO-1 agonist; 50 mg/kg; 51280; Sigma-Aldrich) [36] and Znpp (HO-1 inhibitor; 3 mg/kg; 282820; Sigma-Aldrich) [37] were administered after dissolving in 10% dimethyl sulfoxide (DMSO). EX-527 (5 µg/10 µl/rat; E7034; Sigma-Aldrich) [38] was dissolved in 10% DMSO and injected intrathecally (i.t.).

Experimental design

To assess whether the HO-1/SIRT1 signaling pathway plays an important role in CYP-induced cystitis in rats, we designed the experiments in four parts.

First, we performed a time-gradient experiment to determine associations between HO-1 and neuroinflammation in rats. In this experiment, the rats were divided into five groups (n = 6/group): control, CYP d8, CYP d13, CYP d16, and CYP d19. The control group received an i.p. injection of 0.9% normal saline, and the other groups received i.p. injections of CYP, followed by sampling on days 8, 13, 16, and 19. Different initial CYP administrations were tested in the non-control groups to ensure the same sampling time. All groups were analyzed using behavioral and urodynamic tests. Additionally, we assessed changes in the expression of HO-1 and neuroinflammation-related markers, including glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule-1 (Iba-1), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 by western blot analysis.

To further investigate the involvement of HO-1 in suppressing CYP-induced cystitis, we divided the experiments into three parts. In part 1, we upregulated HO-1 expression in all groups (n = 6/group), including the control, CYP group, CYP + 10%DMSO group, and CYP + Hemin group (rats received i.p. injection of hemin 1 day prior to each CYP injection). In part 2, we randomly assigned animals to four groups (n = 6/group): control, CYP, CYP + 10%DMSO, and CYP + Znpp (rats received i.p. injection of Znpp 1 day prior to each CYP injection). In part 3, rats were divided into four groups (n = 6/group) to verify whether EX-527 (a selective inhibitor of SIRT1) attenuated the efficacy of hemin: control, CYP + 10%DMSO, CYP + Hemin, and CYP + Hemin + EX-527 (rats received i.t. injection of EX-527 once daily for 3 consecutive days starting 1 day after the final CYP injection).

Samples from all groups were harvested on day 13 after the first CYP injection, and behavioral and urodynamic tests and western blot analyses were performed to assess the effects of the intervention on the HO-1/SIRT1 pathway. All experiments were performed in triplicate for each experimental group.

Assessments of mechanical pain

We used mechanical pain as assessed using the mechanical withdrawal threshold to evaluate hyperalgesia. We adopted the up-down method, in which a series of von Frey filaments (rated at 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, and 15 g) were used to stimulate the skin of the lower abdomen of rats in order to measure the mechanical withdrawal threshold [39]. The behavioral test was performed every 3 days starting from the first CYP injection. Positive behavior was considered when the animal licked or scratched the stimulation area or an “intentional dodge”.

Urodynamic test

We performed urodynamic evaluations on the rats to determine periodic pressure changes in the bladder, as previously described [40], which was then used to calculate the micturition interval. At 30 min before the urodynamic test, rats were deeply anesthetized, and a gentle massage was performed on the bladder to expel the urine. A lubricated epidural catheter was then inserted into the bladder cavity through the urethra, and upon reaching the bottom of the bladder, the catheter was pulled 1-cm back. Saline was infused at a rate of 6 mL/h at the other end of the catheter. Changes in bladder pressure were recorded using a urodynamic device (BL- 420F; Taimeng Technology, Shenzhen, China).

Intrathecal injection

Intrathecal injection into rats was performed as previously described [41]. First, the rats were mildly anesthetized with isoflurane. Following sterilization with 75% alcohol, the intervertebral space between L5 and L6 was inserted using a reagent-carrying microinjector, with tail flicking or quivering indicating a successful puncture. After leaving the intervertebral space for 15 s, the needle was withdrawn slowly, and the skin was subsequently disinfected using alcohol.

Western blot

The rats were anesthetized with Ulatan (20%, 1.6g/kg, i.p.) and sacrificed. Following spinal cord harvesting from L6-S1, the SDH was separated and stored at − 80 ℃ until further analysis. Samples were lysed in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. After lysis, the homogenates were centrifuged at 14,800g for 20 min, and the supernatants were harvested and loaded onto 10% sodium dodecyl sulfate polyacrylamide gels after determination of protein concentrations using a bicinchoninic acid protein assay for electrophoresis at 80 V for 30 min and 120 V for 60 min. Proteins in the bands were then transferred onto polyvinylidene fluoride membranes (at 300 mA), which were blocked with 5% skim milk for 1 h at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: HO-1 (1:1000; ab13243; Abcam, Cambridge, UK), SIRT1 (1:1000; ab189494; Abcam), GFAP (1:1000; 3670; Cell Signaling Technology, Danvers, MA, USA), Iba-1 (1:1000; ab5076; Abcam), phosphorylated (p)-c-Jun N-terminal kinase (JNK; 1:1000; 9251s; Cell Signaling Technology), p-p38 (1:1000; 4511; Cell Signaling Technology), IL-1β (1:1000; 16806-1-AP; Proteintech, Rosemont, IL, USA), IL-6 (1:1000; DF6087; Affinity Biologicals, Ancaster, ON, Canada), TNF-α (1:1000; BS1857; Bioworld Technology, Bloomington, MN, USA), and β-actin (1:1000; af7018; Affinity Biologicals). After washing three times with Tris-buffered saline with Tween-20 (TBST), the membranes were incubated at room temperature with secondary antibodies (conjugated with horseradish peroxidase) for 1 h, followed by another three washes with TBST. The immune complexes were then detected using an enhanced chemiluminescence kit (WBKLS0100, Millipore, Billerica, MA, USA), and band intensities were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Immunofluorescence

The rats were perfused transcardially with saline, followed by 4% paraformaldehyde, under deep anesthesia with sodium pentobarbital (50 mg/kg, i.p.). The spinal cord segments (L6-S1) were carefully removed and immersed in 4% paraformaldehyde for fixation after harvesting. After 30 min, spinal cord segments were immersed in a solution of 30% sucrose for dehydration. The spinal cords were then rapidly frozen and cut transversely into 16-µm sections using a freezing microtome. Each section was blocked with immunofluorescent blocking agent for 1 h after three washes with phosphate-buffered saline (PBS) and then incubated with the following primary antibodies overnight at 4°C: HO-1 (1:500; ab13243; Abcam), SIRT1 (1:100; ab189494; Abcam), neuronal nuclei (NeuN; 1:200; MAB377; Millipore), GFAP (1:400; 3670; Cell Signaling Technology), and the CD11b antibody (OX-42; 1:50; MCA275G; Bio-Rad, Hercules, CA, USA). On the next day, after another three washes with PBS, sections were incubated with fluorescein isothiocyanate-conjugated or Cy3-conjugated secondary antibodies for 1 h at 37°C. Nikon fluorescence microscopy (Nikon, Tokyo, Japan) was used to capture images, which were analyzed using ImageJ software.

Statistical analysis

All statistical analyses were performed using SPSS (v.25.0; IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard error of the mean. Student’s t test was used for comparisons involving only two groups (western blot and urodynamic data analysis). Comparisons involving mechanical withdrawal threshold data were performed using two-way analysis of variance, followed by Sidak's multiple comparisons test. A P < 0.05 was considered statistically significant.

HO-1 level is downregulated in the SDH of rats with CYP-induced cystitis and accompanied by glial cell-mediated neuroinflammation

Figure 1A shows western blot results indicating that HO-1 in the CYP-induced cystitis group was downregulated on days 8, 13, and 19 after the first CYP injection relative to levels in the control group, and that the minimum HO-1 expression was achieved on day 13. Additionally, immunofluorescence staining revealed a substantial decrease in HO-1 expression in the SDH of rats with cystitis (Fig. 1B), with double immunofluorescence staining to evaluate localization revealing that the majority of HO-1 was mainly co-localized with NeuN and the remainder in astrocytes and microglia (Fig. 1C).

Additionally, von Frey filament tests displayed (Fig. 2C) the pain threshold initial decreases, followed by increases that reaches a trough point on day 13. Urodynamic examinations to record changes in intravesical pressure, which indirectly reflect the voiding interval of rats after CYP injection, revealed a more severe urinary frequency in the CYP group relative to the control group (Fig. 2A and B). Meanwhile, we also found that GFAP and Iba-1 (markers of astrocytes and microglia, respectively) expression was respectively upregulated (Fig. 2D and E). Moreover, we found that IL-1β, IL-6, and TNF-α levels increased significantly in the cystitis group relative to those in the control group (Fig. 2F–H). These results suggested that CYP injection downregulated HO-1 level, and that HO-1 might play a prominent role in neuroinflammation and mechanical hyperalgesia in the cystitis model.

Upregulating HO-1 attenuates mechanical allodynia and urinary frequency in cystitis-model rats

We then administered the HO-1 agonist hemin to determine a potential connection between HO-1 and cystitis-related symptoms. Hemin was administered 1 day before each CYP injection. Assessment of changes in mechanical pain and bladder pressure showed no statistical differences in micturition intervals between CYP and CYP + DMSO groups, while urinary frequency was significantly attenuated after hemin treatment relative to the CYP + DMSO group (Fig. 3A and B). Additionally, the von Frey filament test also indicated a difference between the CYP + Hemin and CYP + DMSO groups on day 4 after the first CYP injection, with this significant difference persisting > 10 days (Fig. 3C).

HO-1 upregulation promotes SIRT1 expression and alleviates neuroinflammation in CYP-induced cystitis-model rats

To assess the effect of HO-1 upregulation on neuroinflammation in the cystitis-model rats, we performed western blot analysis of samples harvested on day 13 after the first CYP injection. We found that hemin injection increased HO-1 and SIRT1 expression (Fig. 4A and B), with levels significantly higher than those in the CYP + DMSO group. Additionally, hemin alleviated neuroinflammation in cystitis-model rats based on the observed downregulation of IL-1β, IL-6, and TNF-α levels in the CYP + Hemin group relative to those in the CYP + DMSO group (Fig. 4E–G). Furthermore, measurement of p-JNK and p-p38 confirmed phosphorylation levels of JNK/p38 decreased after hemin treatment (Fig. 4C and D).

HO-1 inhibition aggravates mechanical allodynia and urinary frequency in cystitis-model rats

To further validate the role of HO-1 in rats with cystitis, we evaluated whether HO-1 inhibition exacerbated cystitis symptoms. Znpp, a competitive HO-1 inhibitor, can compete with heme and thereby inhibit HO-1 activity [42]. We found that following i.p. injection of Znpp on the day before each CYP administration, urodynamic examination of rats for 13 days after the first CYP injection revealed a shorter voiding interval in the CYP + Znpp group relative to that in the CYP + DMSO group, suggesting that the symptoms of urinary frequency became more severe (Fig. 5A and B). Additionally, the mechanical threshold in the CYP + Znpp group from days 4 to 19 was consistently lower than that in the CYP + DMSO group (Fig. 5C).

Neuroinflammation in CYP-induced cystitis-model rats is further aggravated by HO-1 inhibition

Western blot analysis revealed that HO-1 and SIRT1 expression was significantly downregulated after Znpp injection (Fig. 6A and B), whereas p-JNK and p-p38 levels in the SDH in the CYP + Znpp group were elevated relative to those in DMSO-treated cystitis-model rats (Fig. 6C and D). Additionally, analysis of inflammatory factor expression showed that IL-1β, IL-6, and TNF-α levels increased following Znpp injection (Fig. 6E–G). These results confirmed that HO-1 inhibited astrocyte and microglia activation and alleviated neuroinflammation, mechanical allodynia, and urinary frequency in cystitis-model rats. Moreover, they indicated that SIRT1 expression might be positively affected by HO-1 activation.

SIRT1 is critical for the inhibitory effect of HO-1 on mechanical allodynia and urinary frequency in rats with cystitis

To confirm the association between HO-1 and SIRT1, we performed co-localization analysis using immunofluorescence staining of SIRT1 and HO-1. Figure 7 shows that SIRT1 was mainly expressed in neurons, whereas a limited amount was observed in glial cells, which was similar to HO-1 localization. Moreover, we evaluated the therapeutic effects of SIRT1 inhibitors following HO-1 agonist treatment. We found that i.t. injection of EX-527, a specific inhibitor of SIRT1 [38, 43], for 3 consecutive days at 2 days after the third CYP injection exacerbated the urinary frequency of cystitis-model rats following hemin treatment (Fig. 8A and B). Additionally, compared with the CYP + Hemin group, EX-527 attenuated increases in the mechanical withdrawal threshold and the highest peak on day 19 (Fig. 8C).

HO-1/SIRT1 signaling inhibits neuroinflammation in CYP-induced cystitis-model rats by inhibiting JNK/p38 signaling

To investigate the role of SIRT1 in the anti-inflammatory effects of HO-1 in the cystitis-model rats, we performed western blot to further analyze levels of SIRT1, p-JNK/p-p38, and inflammatory factors in the SDH after EX-527 treatment. Figure 9A shows that EX-527 treatment downregulated SIRT1 expression relative to that in CYP + Hemin group. Additionally, compared with the CYP + Hemin group, p-JNK and p-p38 levels increased, as expected, in the CYP + Hemin + EX-527 group (Fig. 9B and C). Moreover, we found that levels of IL-1β, IL-6, and TNF-α correspondingly increased after EX-527 administration (Fig. 9D–F). These data suggested that HO-1 might promote SIRT1 expression to suppress activation of JNK/p38 signaling and reduce the release of IL-1β, IL-6, and TNF-α.

In this study, we identified a novel HO-1–SIRT1–JNK/p38 signaling axis associated with the inhibition of neuroinflammation in rats with CYP-induced cystitis (Fig. 10). We found that HO-1 expression was downregulated in the SDH of cystitis-model rats, and that HO-1 upregulation could activate SIRT1 and significantly alleviate neuroinflammation, mechanical allodynia, and urinary frequency in rats with cystitis. Moreover, the results demonstrated that the therapeutic effect of HO-1 upregulation on cystitis-related symptoms can be reversed by SIRT1 inhibition.

HO-1 is the only isoform that plays a crucial role in cytoprotection against oxidative stress, inflammation, and other aggravating factors [44]. The inhibitory function of HO-1 against the inflammatory response simultaneously includes an analgesic effect, which has been reported in multiple disease models [45–47]. IC/BPS involves chronic inflammation of the bladder and is accompanied by constant pain. Consistently, therapies aimed at the bladder show poor efficacy, because the mechanisms that underlie IC pathogenesis are yet to be fully explained. We previously proposed that the pathogenesis of CYP-induced cystitis might be explained by spinal neuroinflammation [8]. When the bladder epithelium is pressed by an external stimulus, sensory signals from the bladder are carried to the SDH through the afferent nerve, the endings of which release excitatory neurotransmitters, which then activate surrounding glial cells to release proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α. These cytokines mediate neuroinflammatory reactions and contribute to synaptic plasticity, eventually causing hyperalgesia [48, 49].

We found that HO-1 downregulation was accompanied by the activation of glial cells and overexpression of proinflammatory factors in the SDH of cystitis-model rats. To understand the potential effect of HO-1 on spinal neuroinflammation in CYP-induced cystitis, we examined how HO-1 upregulation and downregulation affects cystitis-related symptoms mediated by glial cell activation. We found that the symptoms in rats were alleviated after HO-1 induction but aggravated after HO-1 inhibition. Moreover, several studies report the anti-inflammatory effects of HO-1. Shen et al. [50] found that HO-1 induction inhibits glial-mediated neuroinflammation in the spinal cord to attenuate vincristine-induced neuropathic pain. Even in the IC/BPS model, Hmox1 mRNA expression was identified in bladder tissue, and induction of HO-1 can significantly alleviate bladder inflammation and decrease bladder wet weight [36]. This evidence suggests that HO-1 might attenuate neuroinflammation in rats with cystitis.

The mitogen-activated protein kinase (MAPK) pathway regulates inflammatory gene expression, and p38 and JNK play crucial roles in the release of proinflammatory cytokines in microglia and astrocytes [51]. A study showed that p38 in microglia may mediate neuroinflammation and result in TNF-α and IL-1β overexpression by activating MAPK-activated protein kinase 2 and mitogen- and stress-activated kinase 1 [52]. Other studies showed that JNK phosphorylation in astrocytes is markedly increased during stress-induced hyperalgesia [53], and that inhibition of JNK signaling can mitigate neuroinflammation, which appears to be neuroprotective [54]. In the present study, we demonstrated that HO-1 could alleviate neuroinflammation in rats with cystitis by inhibiting activation of the JNK/p38 signaling pathway. Song et al. [55] suggested that HO-1 could protect neurons against brain ischemic injury by downregulating the JNK signaling pathway, and Huang et al. [56] found that curcumin attenuated PM2.5-induced oxidative stress and inflammatory damage in mice with lung injury through the HO-1/CO/p38 MAPK pathway.

Among sirtuins, SIRT1 is the most extensively studied. SIRT1 has various regulatory functions that can catalyze the deacetylation of histones and non-histone substrates [57, 58]. Accumulating evidence suggests that SIRT1 plays a role in neuroinflammation, with one study indicating that SIRT1 activation significantly reduces the release of TNF-α and IL-6 in microglial BV2 cells after lipopolysaccharide stimulation [59]. Additionally a previous report found that SIRT1 inhibition led to upregulated TNF-α and IL-1β expression following astrocyte activation [60]. Moreover, SIRT1 reportedly reduces neuroinflammation via the MAPK pathway, as SIRT1 overexpression significantly reduces activation of JNK/p38 signaling, thereby improving neurological dysfunction or death induced by the overproduction of inflammatory cytokines after traumatic brain injury [61]. Similarly, Yang et al. [26] demonstrated that SIRT1 activation might exert a neuroprotective effect through the p38 MAPK pathway. In the present study, we found that SIRT1 inhibition upregulated levels of p-JNK and p-p38 in cystitis rats. These findings indicate that SIRT1 might suppress neuroinflammation in CYP-induced cystitis by regulating the JNK/p38 signaling pathway.

SIRT1 is widely expressed in the CNS. Ramadori et al. [23] found that SIRT1 mRNA was mainly expressed in neurons in the hypothalamus and cerebellum. In the present study, co-localization analysis via immunofluorescent staining revealed that SIRT1 in the SDH was predominantly expressed in neurons, providing evidence for its interaction with HO-1. The anti-inflammatory effect exerted by HO-1 is generally achieved through the production of its enzymatic byproduct CO [62], whereas SIRT1 can act as a downstream target of CO. A previous study showed that CO alleviates 5FU-induced endothelial senescence through SIRT1 activation [63]. Additionally, Park et al. [64] suggested that CO can significantly enhance SIRT1 expression, which is crucial for anti-aging and antioxidant effects through downregulation of microRNA-34a. Moreover, previous reports highlighted a role for the HO-1/SIRT1 signaling pathway in regulating inflammation. Specifically, myeloid-specific HO-1 deletion exacerbated liver inflammation due to a lack of SIRT1, whereas SIRT1 activation improved the anti-inflammatory function in HO-1-deficient macrophages [65]. Furthermore, Liu et al. [66] reported that cobalt protoporphyrin, an inducer of HO-1 activity, upregulated SIRT1 expression in C57BL/6J mice fed a high-fat diet and ameliorated liver metabolic damage. In the present study, we found that changes in HO-1 expression positively affected SIRT1 activation. Further in vivo experiments to verify the role of the HO-1/SIRT1 pathway in rats with cystitis involved EX-527 administration to antagonize SIRT1 specifically after hemin injection, which revealed that the therapeutic effect of HO-1 upregulation after hemin treatment could be reversed by SIRT1 inhibition.

This study has limitations. Although we demonstrated the important role of the HO-1/SIRT1 signaling pathway in attenuating cystitis-related symptoms via inhibition of glial cell activation, the detailed molecular regulatory mechanism between SIRT1 and the JNK/p38 pathway remains unclear, and the mechanism by which HO-1 activates SIRT1 requires further investigation. Additionally, whether there is an underlying interaction between microglia and astrocytes after activation needs to be elucidated by additional research.

This study demonstrated that SIRT1 might mediate the suppressive effect of HO-1 on cystitis-related symptoms in CYP-induced cystitis-model rats by inhibiting JNK/p38 pathway-induced neuroinflammation in the SDH. Therefore, HO-1 upregulation might represent a promising therapeutic target for the treatment of cystitis.

BPS

bladder pain syndrome

carbon monoxide

CYP

cyclophosphamide

CNS

central nervous system

DMSO

dimethyl sulfoxide

EPC

endothelial progenitor cell

GFAP

glial fibrillary acidic protein

high glucose

HO-1

heme oxtygenase-1

i.p.

intraperitoneal

i.t.

intrathecal

Iba-1

ionized calcium-binding adapter molecule-1

interstitial cystitis

interleukin

JNK

c-Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

NAD

nicotinamide adenine dinucleotide

SDH

spinal dorsal horn

SIRT1

silent information regulator 1

TNF

tumor necrosis factor

Znpp

Protoporphyrin IX zinc(II)

Acknowledgments

Not applicable

Authors’ contributions

ZTG designed and performed most of the experiments, analyzed and interpreted the data, and wrote the manuscript. QQG and YH participated in animal experiments and contributed to critical manuscript revision. MLL and CZ participated in data analysis. MZS and JLC helped with data collection. HLZ and BLL conceived and supervised this study. BLL and XFZ provided funding and revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (82170786, 81800666, 82100816); the Natural Science Foundation of Guangdong Province (2022A1515010250, 2021A1515010354); the Guangzhou Science and Technology Program key project (202103000035); the Cultivation Special Project Foundation of The Third Affiliated Hospital of Sun Yat-Sen University (2022GZRPYQN07)；the Guangdong Basic and Applied Basic Research Foundation (2021A1515111199).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

All experiments were approved by the Institutional Animal Care and Use Committee at the Sun Yat-Sen University and performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85–23; revised 1985).

Consent for publication

All authors agree to the publication of this manuscript.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Urology, The Third Affiliated Hospital and Lingnan Hospital of the Sun Yat-sen University, 2693 Kaichuang Road, Guangzhou 510700, China.

²Department of Rehabilitation, The Third Affiliated Hospital and Lingnan Hospital of the Sun Yat-sen University, 2693 Kaichuang Road, Guangzhou 510700, China.

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No competing interests reported.

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HO-1 alleviates neuroinflammation and cystitis-related symptoms in cyclophosphamide-induced cystitis via the SIRT1 pathway

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Animals

Reagents

Experimental design

Assessments of mechanical pain

Urodynamic test

Intrathecal injection

Western blot

Immunofluorescence

Statistical analysis

Results

HO-1 level is downregulated in the SDH of rats with CYP-induced cystitis and accompanied by glial cell-mediated neuroinflammation

Upregulating HO-1 attenuates mechanical allodynia and urinary frequency in cystitis-model rats

HO-1 upregulation promotes SIRT1 expression and alleviates neuroinflammation in CYP-induced cystitis-model rats

HO-1 inhibition aggravates mechanical allodynia and urinary frequency in cystitis-model rats

Neuroinflammation in CYP-induced cystitis-model rats is further aggravated by HO-1 inhibition

SIRT1 is critical for the inhibitory effect of HO-1 on mechanical allodynia and urinary frequency in rats with cystitis

HO-1/SIRT1 signaling inhibits neuroinflammation in CYP-induced cystitis-model rats by inhibiting JNK/p38 signaling

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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