Diacerein modulates TLR4/ NF‐κB/IL‐1β and TRPC1/CHOP signalling pathways in gentamicin‐induced parotid toxicity in rats

Abstract The present study aimed to identify the possible protective effect of diacerein (DIA) on gentamicin (GNT)‐induced parotid toxicity in rats. DIA was administered in the presence and absence of GNT. Thirty‐two Wistar adult male rats were randomly arranged into four groups: control, DIA (50 mg/kg/day), GNT (100 mg/kg) and GNT+DIA groups for 8 days. Parotid oxidative stress parameters, besides inflammatory and apoptotic biomarkers, were evaluated. Salivary flow rate, transient receptor potential canonical 1 (TRCP1), and C/EBP homologous protein (CHOP) in parotid tissue were measured. A parotid histopathological examination and an interleukin‐1 beta (IL‐1β) immunohistochemical study were also performed. GNT significantly increased parotid oxidative stress, inflammatory, apoptotic and CHOP biomarkers with decreased salivary flow rate and TRCP1 level. A histopathological picture of parotid damage and high IL‐1β immunoexpression were detected. DIA significantly normalized the distributed oxidative, inflammatory and apoptotic indicators, CHOP and TRCP1, with a prompt improvement in the histopathological picture and a decrease in IL‐1β immunoexpression. These results reported that DIA protects against GNT‐induced parotid toxicity via modulation of TLR4/NF‐κB/IL‐1β and TRPC1/CHOP signalling pathways.


| INTRODUC TI ON
Gentamicin (GNT), a type of aminoglycoside, is considered the most vital antibiotic agent against a variety of species, particularly those that are resistant to other antibacterials. It is operative against gram-negative and inadequate gram-positive organisms. It is among the earliest and best-known remedies for a number of bacterial diseases-such as bone, chest and urinary tract infections-pelvic inflammatory diseases, meningitis and sepsis. GNT is regarded by the WHO as being of vital importance for human medicine and is listed as one of the essential medicines. 1 It functions by impeding the bacterial capacity to produce proteins, which usually results in bacterial death. Before being excreted in the kidneys via glomerular filtration, GNT is transported basically unaltered throughout the extracellular space instead of being metabolized. Potentially harmful side effects, most frequently ototoxicity 2 and nephrotoxicity, 3 place restrictions on its use. According to Abdollahi et al., 4 GNT may have an impact on the salivary glands.
In humans, there are 10 members of the large family of transmembrane recognition proteins known as Toll-like receptors (TLR). It is crucial to the detection of pathogens and the activation of innate immunity. They are capable of identifying the pathogen-associated molecular patterns that are expressed on infectious pathogens and facilitating the synthesis of the cytokines required for the maturation of functional immunity. TLR4 has recently been shown to be crucial for triggering the inflammatory response. When it is activated, an intracellular signalling pathway of proinflammatory cytokines, such as nuclear factor-kappa B (NF-κB), tumour necrosis factor (TNF), and interleukin-1 (IL-1), is stimulated. 5 The human transient receptor potential canonical or classical (TRPC1) protein comprises functional nonselective cationic channels with high calcium permeability. These channels are widely expressed in different human tissues and cell types. By supplying the Ca 2+ influx pathway or depolarizing the membrane potential, they are an important factor in the control of intracellular calcium. 6 These ion channels are therefore prospective targets for drugs to treat diseases like cancer, epilepsy, pain, arthritis and cardiac remodelling. 7 The acinar and ductal cells of the salivary gland are thought to secrete fluid mostly in response to TRPC1. When TRPC1 is damaged, the endoplasmic reticulum (ER) Ca 2+ level drops, salivary gland cells die and C/EBP homologous protein (CHOP) expression rises. 8 Diacerein (DIA), a novel analgesic, antipyretic and antiinflammatory medication created specifically for the treatment of osteoarthritis, is a derivative of anthraquinone. DIA works by preventing human monocytes from producing IL-1. By triggering NF-κB and mitogen-activated protein kinase signalling, interleukin-1, a pro-inflammatory and pro-apoptotic substance, stimulates the production of cytokines. DIA has been shown to significantly reduce inflammatory damage by downregulating the interleukin-1 beta (IL-1β) receptor, 9 as well as inhibiting the production of nitric oxide inflammatory cytokines such as IL-1β and TNFα and preventing the TLR4/NF-κB-mediated signalling pathways. 10 Salivary glands have become a valuable research tool to investigate some fundamental issues in physiology, pathology and pharmacology, including protein synthesis, salt and water transport and autonomic nerves and receptor pharmacology. 11 Even though GNT is frequently used in anti-infectious therapy, its negative effects on the salivary glands have not received enough attention. As a result, we were curious to explore the potential protective role of DIA against the effects of GNT, a commonly used antibiotic, on the rat parotid glands, with a focus on the TRPC1/CHOP and TLR4 signalling pathways, as well as the likely mechanisms mediating its impact.

| Ethics
Rats' processing and therapy were done in accordance with the Institutional Ethical Committee's (Faculty of Medicine, Minia University, Egypt) guidelines for the care of experimental animals, as well as the National Institutes of Health's (NIH) Guide for the care and use of laboratory animals (Approval number: 398:2022).

| Animals and experimental design
Thirty-two male Wistar rats weighing 220-250 g, aged 7-10 weeks, were acquired from the National Research Center. Rats were left for acclimatization in their cages (4 rats/cage) in a normal light-dark cycle

| Sample collection
At the end of the experiment, the salivary flow rate was determined.
Under the effect of anaesthesia with urethane hydrochloride (1 g/ kg i.p.), salivation was stimulated by pilocarpine (1.0 mg/Kg body weight, i.p.), which was dissolved in distilled water, and the saliva that was produced was dripped in plastic tubes and maintained on ice during all the saliva collection period (40 min). 14 Then, rats were sacrificed, and blood was collected from the abdominal aorta of the rats in heparinized syringes. It was then centrifuged at 4000 × g for 15 min (Janetzki T30 centrifuge). Then, sera were kept at −80°C for biochemical analysis. The parotid glands were excised and rinsed with saline to eliminate any blood. A part was fixed for histological studies. The other part was homogenized in ice-cold phosphate buffer (0.01 M, pH 7.4; 20% w/v) with a glass homogenizer on ice (tissue weight (g): phosphate buffer (mL) volume = 1:5). The homogenate was centrifuged for 15 min at 5000 rpm, and the supernatant was stored at −80°C to measure the biochemical parameters.

| Assessment of oxidative stress parameters
Malondialdehyde (MDA) is an index of lipid peroxidation. It was measured using the thiobarbituric acid method, and the sample absorption was determined at 535 nm. 15 According to the previously prescribed method of Marklund and Marklund, 16 one unit of superoxide dismutase (SOD) is equal to the amount of enzyme that prevents pyrogallol autoxidation by 50%. A spectrophotometer was used to measure enzyme activity at 420 nm.
TAC was estimated by colorimetric kits according to the manufacturer's instructions.

| Assessment of TRCP1 and CHOP levels
Parotid TRCP1 and CHOP levels were measured using ELISA kits, following the manufacturer's instructions. In CHOP, the colour of the tubes changed from blue to yellow, and the optical density was read at 450 nm within 15 min. In TRCP1, the colour turned yellow immediately, and the optical density was read at 450 nm after adding the stop solution.

| Assessment of inflammatory and apoptotic parameters
TNF-, NF-B, and caspase-3 levels were measured using ELISA kits as directed by the manufacturer.

| Western blotting of TLR4 and cleaved caspase-3
Regarding western blotting, parotid gland homogenates (30 μg of total proteins) were heated for 5 min with loading buffer, then placed on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which was then exposed to running at 100 V for 2 h.

| Histological study
Specimens from the parotid gland were fixed, then dehydrated, cleared and embedded in paraffin. Sections with a thickness of 5 μm were cut and stained. Sections were examined blindly. The inflammation was scored from 0 to 3. A score of 0 indicates no inflammation; a score of 1 indicates mild inflammation with no foci; a score of 2 indicates moderate inflammation and one focus. A score of 3 indicates severe inflammation and more than one focus. 17

| Immunohistochemical staining
Immunohistochemistry for IL-1β was performed on paraffinembedded tissue according to the manufacturer's protocol.

| Statistical analysis
All values were expressed as mean ± SEM. The statistical analyses were conducted using GraphPad Prism (version 5.0). Analysis of variance (anova) for multiple comparisons, followed by the Tukey-Kramer test as a postanova test, was used for data analysis. The results were considered statistically significant if p < 0.05.

| Effect of DIA on biochemical parameters in GNT-induced parotid toxicity in rats
The GNT group showed significantly decreased parotid SOD and serum TAC with increased parotid MDA when compared to the control and DIA groups. Meanwhile, the GNT+DIA co-treated rats showed significantly improved oxidative stress parameters when compared to the GNT group ( Table 1).
In Figure 1, rats challenged with GNT revealed a significant decrease in salivary flow rate and parotid TRCP1 level and a significant increase in parotid CHOP level in comparison to the control and DIA groups. The GNT+DIA co-treated rats showed a significant increase in salivary flow rate and parotid TRCP1 level and a significant decrease in parotid CHOP level when compared to the GNT group.
In Table 2, parotid TNFα, NF-κB, and caspase-3 levels significantly increased in the GNT group when compared to the control and DIA groups. On the contrary, the GNT+DIA group showed a significant decrease in the level of parotid TNFα, NF-κB and caspase-3 when compared to the GNT group.
The GNT group showed a significant increase in parotid TLR4 and C caspase-3 expressions when compared to the control and DIA groups. Meanwhile, when compared to the GNT group, the GNT+DIA co-treated rats had a significant decrease in parotid TLR4 and C caspase-3 expressions (Figure 2A,B). The mean value of the inflammation score was expressed as mean ± SEM (8 rats/group).

TA B L E 1 Effect of DIA on oxidative stress parameters in GNT-induced parotid toxicity in rats.
the treatment and prevention of such infections. Gentamycin is an aminoglycoside antibiotic derived from Micromonospora purpurea and is used in the treatment of many types of bacterial infections, particularly those caused by Gram-negative organisms. 18 Despite its wide clinical use, gentamycin has been reported to cause disturbing toxicity. One of the effects of this toxicity is the effect of GNT on salivary glands. 19 The findings of this study revealed that GNT caused parotid tox-   IL-6 and TNFα. 33 On the contrary, NF-κB is a key regulator of the production of numerous immunomodulatory mediators connected to oxidative stress. 34 The oxidative instability that increases the outflow of proinflammatory cytokines by activating the redox-sensitive transcription factor is related to the inflammatory response of GNT  Open Access funding enabled and organized by Projekt DEAL.

FU N D I N G I N FO R M ATI O N
This work was supported by the University of Witten-Herdecke Germany.