Introduction

Inflammation has become a global target for scientific research because of its implication in multiple human diseases (Siebert et al. 2015). Inflammation is a complex biological response of vascular tissues to various harmful stimuli such as pathogens, antigens and toxic chemicals. Chronic inflammatory joint disease like rheumatoid arthritis (RA) is an exaggerated immune response to self-antigen (Jia et al. 2016). RA involves a complex interplay among inflammation and pain (Amarasekara et al. 2015). Macrophages activated by TLR and NLR via interaction with endogenous or exogenous ligands are responsible for the expression of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF-α) and enzymes that mediate activation and differentiation of leukocytes, endothelial cells and chondrocytes (Brennan et al. 2002; Hess et al. 2011). These mediators contribute to the expression of endothelial adhesion molecules, protection of synovial fibroblast, promotion of angiogenesis and suppression of regulatory T cells and pain induction (Brennan et al. 2002; Hess et al. 2011). Transgenic mice over expressing TNF-α exhibit a chronic inflammatory and rapidly destructive arthritis (Keffer et al. 1991). In several other studies of animal model of experimental chronic inflammation supported that IL-1 (Koenders et al. 2005) and IL-6 deficient mice show a diminished cartilage damage, bone resorption and synovial immune infiltrate (Jia et al. 2016). These cytokines have diversed site and mechanism of action as well as involve in sensitization and activation of nociceptors (Spurlock et al. 2014). Chronic inflammation is associated with chronic pain that causes plastic changes in neurons of peripheral and central nervous system and characterized by exaggerated painful response to noxious (hyperalgesia), innocuous (allodynia) stimulation along with persistent pain at rest (arthritis pain) (Hernstadt et al. 2009). In inflammatory pain condition, pro-inflammatory cytokines (IL-1, IL-6, TNF-α) and various small molecules (e.g., ATP, bradykinines, prostaglandins) recruit towards site of injury and act on nociceptors leading to hyperalgesia, allodynia and spontaneous pain (Khan et al. 2014).

Currently, conventional disease-modifying, non-steroidal anti-inflammatory drugs and various other corticosteroids are being used for the treatment of inflammatory diseases. These therapies sometimes fail or produce some partial responses and their serious side effects and low efficacy limit the use of these agents (Gibofsky 2014). Therefore, in search of new most effective and safe anti-inflammatory agent, MOPBS was evaluated in various models of inflammation. Previously, antiviral and cytostatic activity of 2-(5-mercapto-1,3,4-oxadiazol -2-yl)-N-propylbenzenesulphonamide was investigated on several viral strain (such as cytomegalovirus, varicella-zoster virus) and on proliferation of murine leukemia cell (L1210), and cervix carcinoma (HeLa), humanT-lymphocyte cells (CEM) (El-Sabbagh 2013; Syed et al. 2011). Based on previous findings, 2-(5-mercapto-1,3,4-oxadiazole-2-yl)-N-propylbenzenesulphonamide was synthesized, starting from sodium saccharin (El-Sabbagh 2013) and its pharmacological activity was evaluated by designing carrageenan and CFA-induced inflammatory mice models.

Materials and methods

Chemicals and reagent

MOPBS (Fig. 1a) was synthesized in the synthetic Organic Chemistry laboratory at Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad Pakistan and was mainly characterized by FTIR, NMR and Elemental analysis. Carrageenan, Complete Freundˈs Adjuvant, Griess reagent, Acetone, Piroxicam, and Dexamethasone were obtained from Sigma (USA). Trizol reagent was obtained from Witrogen (USA). All the above-mentioned drugs were dissolved and diluted in normal saline and 2% DMSO.

Fig. 1
figure 1

a Structure of 2-(5-mercapto-1,3,4-oxadiazol-2-yl)-N-propylbenzenesulphonamide (MOPBS). b MOPBS (1, 10, 50 and 100 mg/kg) pretreatment reduced carrageenan-induced paw oedema. The paw thickness was measured after 4 h of carrageenan injection. c MOPBS (1, 10, 50 and 100 mg/kg) pretreatment inhibited carrageenan-induced mechanical hyperalgesia, d mechanical allodynia and e thermal hyperalgesia. The paw withdrawal effect was measured after 4 h of carrageenan injection The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 show a significant difference from carrageenan-induced group. (###) denotes comparison of vehicle control group (normal group) and carrageenan-induced group with MOPBS-treated group

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Physicochemical properties of 2-(5-mercapto-1,3,4-oxadiazol-2-yl)-N-propylbenzenesulphonamide (MOPBS)

Off-white solid, Mol.Formula: C11H13N3O3S2, Mol. Wt.: 299.36 g/mol, m.p 140–142 °C, yield 65%, Rf = 0.38, (n-Hexane: EtOAc 8:2), solubility: EtOH, acetone, normal saline and DMSO, (FTIR cm−1): 1652 (C=N), 1076 (C–O–C), 2551 (SH), 3091 (CH–Ar), 3090(NH); 1H-NMR (400 MHz, CDCl3): δ 12.5 (s, 1H, SH), 8.04-8.00 (m, 4H, ArH), 7.74 (s, 1H, NH), 3.50 (t, 2H, J = 6.9 Hz, CH2), 1.60 (sext., 5H, J = 6.5 Hz, CH2), 0.90 (t, 3H, J = 6.8 Hz, CH3); 13C- NMR(400 MHz, CDCl3): δ 165.9, 163.8, 137.8, 133.5., 132.4, 129.0, 127.8, 45.0,22.0, 11.2; Elemental analysis: calcd for C11H13N3O3S2 (299.36) C, 44.13; H, 4.38; N, 14.04, O, 16.03, S, 21.42 found: C, 44.10; H, 4.36; N, 14.10, O, 16.00, S, 21.40.

Animals

Male albino mice (24–26 g), 3–4 weeks of age, were purchased from NIH, Islamabad, Pakistan. All animal experiments were performed in pathogen-free zone of pharmacology laboratory according to the Bioethical committee protocols for Care and Use of laboratory Animal (Quaid-i-Azam University, Islamabad) under Ethical Committee code (Approval No. BES-FB-QAU2014-4/20-06-2017). Animals were kept under standard environmental conditions (23 ± 1 ± 0.05 °C with 10% humidity in a 12 h light–dark cycle) and maintained with free access to standard laboratory diet and water.

Animal models

In carrageenan-induced model, animals were divided into seven groups and each group contained seven animals:

Group I::

Vehicle control animals received saline with 2% DMSO (i.p)

Group II::

Carrageenan-induced group (100 μg/paw, i.pl)

Group III::

Dexamethasone 5 mg/kg (i.p, 1 h before carrageenan administration)

Group IV::

MOPBS 1 mg/kg (i.p, 1 h before carrageenan administration)

Group V::

MOPBS 10 mg/kg (i.p, 1 h before carrageenan administration)

Group VI::

MOPBS 50 mg/kg (i.p, 1 h before carrageenan administration)

Group VII::

MOPBS 100 mg/kg (i.p, 1 h before carrageenan administration)

In CFA-induced model, animals were divided into four groups and each group contained seven animals:

Group I::

Vehicle control animals received saline with 2% DMSO (i.p)

Group II::

CFA-induced group (20 μl/paw, i.pl)

Group III::

Dexamethasone 5 mg/kg (i.p, 40 min before CFA administration)

Group IV::

MOPBS 50 mg/kg (i.p, 40 min before CFA administration)

Animals received (vehicle control) saline with 2% DMSO, dexamethasone 5 mg/kg and MOPBS 50 mg/kg on daily basis and treatment was continued until 21 days.

behavoural experiments

Evaluation of paw oedema in carrageenan and CFA-induced mice

Paw thickness was assessed to determine the effect of MOPBS pretreatment on inflammatory paw oedema. Paw oedema was measured using Dial thickness gauge (No.2046F, Mitutoyo, Kawasaki, Japan). Paw thickness was recorded after 4 h of carrageenan injection (Khan et al. 2013a). In chronic CFA-induced arthritis model, paw oedema was measured before the induction of oedema as baseline and at 2, 4, 6 h at day 1 and then continuing from day 1 up to day 21 (Kang et al. 2008).

Evaluation of mechanical hyperalgesia in carrageenan and CFA-induced mice

Mechanical hyperalgesia was determined using Randall Selitto test (Digital Paw Pressure Randall Selitto Meter, IITC Life Science Inc Woodland Hills, CA) according to the previously reported method (Arora et al. 2015). Briefly, 15–30 min before the start of Randall Selitto test, animals were kept in a quiet room for acclimatization. The tip of force transducer was applied perpendicularly to the central area of the hind paw and pressure was increased gradually until mice showed withdrawal response. The nociceptive response included the removal of paw followed by clear movements. The pressure was recorded automatically when mice showed withdrawal response. The mechanical hyperalgesia was measured after 4 h of carrageenan injection. Similarly, in chronic arthritis model, mechanical hyperalgesia was evaluated before the induction and at 2, 4, 6 h continuing from day 1 up to day 21 after CFA injection.

Evaluation of mechanical allodynia in carrageenan and CFA-induced mice

To investigate the effect of MOPBS, on mechanical allodynia, Von Frey test was performed according to the previously reported methodology (Mohammadi and Christie 2014). Animals were placed in individual chamber of Von Frey mesh table to allow the free access to ventral surface of right hind paw. Mice were placed in each box of Von Frey mesh 1 h prior to behavior test for acclimatization. Paw withdrawal was considered as positive nociceptive response. Each Von Frey filament (Stoelting, USA) was applied for five times with increasing order of force. Withdrawal reflex of at least three out of five applications was considered as positive response. Acute effect of MOPBS on mechanical allodynia was evaluated at 4 h post-carrageenan injection. In chronic arthritis case, mechanical allodynia was evaluated before induction and at 2, 4, 6 h continuing from day 1 up to day 21 after CFA injection.

Evaluation of thermal hyperalgesia in carrageenan and CFA-induced mice using hot plate test

Thermal hyperalgesia was determined using hot plate test reported previously (Bölcskei et al. 2005). The latency time (s) of paw withdrawal was determined until 60 s. Effect of acute pretreatment of MOPBS on thermal hyperalgesia was evaluated at 4 h after carrageenan injection. In chronic arthritis model, thermal hyperalgesia was evaluated before induction and at 2,4,6 h continuing from day 1 up to day 21 after CFA injection (Arora et al. 2015).

Evaluation of cold allodynia in CFA-induced arthritis mice by using cold acetone test

Cold allodynia was assessed using cold acetone test as described previously (Longo et al. 2013).

Biochemical assays

Nitric oxide (NO) determination in CFA-induced blood plasma

To determine the effect of MOPBS 50 mg/kg on NO production in blood plasma, Griess reagent test was performed according to the previously described method (Khan et al. 2011).

Determination of inflammatory cytokines and antioxidant enzymes by quantitative real-time PCR

Paw skin tissues were removed by homogenization and the total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA) and qRT-PCR was conducted as described previously (Khan et al. 2014). Briefly, quantitative real-time (qRT-PCR) analysis for several target genes (TNF-α, IL-1β, IL-6, Nrf2, HO-1, SOD2, VEGF, and β-actin) was performed using Applied Biosystems (AB) detection instruments and software. Forward and reverse primers for each gene were: β-actin, 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′and 5′-CATGTAGGCCATGAGGTCCACCAC-3′, VEGF; 5′-TTACTGCTGTACCTCCACC-3′ and 5′-ACAGGACGGCTTGAAGATG-3′. For SOD2; 5′-GCGGTCGT GTAAACCTCAT-3′ and 5′-GGTGAGGGTGTCAGAGTGT-3′. For HO-1; 5′-CACGCATATACCCGCTACCT-3′ and 5′-CCAGAGTGTTCATTCGAGA-3′, For Nrf2; 5′-TGGGGAACCTGTGCTGAGTCACTGGAG-3′ and 5′-ACCCCTTGGACACGACTCAGTGACCTC-3′. For IL-6; 5′-CCACTTCACAAGTCGGAGGCTTA-3′ and 5′-CCAGTTT CCAGTTTGGTAGCATCCATCATTTC-3′. For IL-1β; 5′-TCCAGGATGAGGACATGAGCAC-3′ and 5′-GAACGTCACCCAGCAGGTTA-3′. For TNF-α; 5′-GTTCTATGGCCCAGACCCTCA-3′ and 5′-GGCACCAC TAGTTGGTTGTCTTTG-3′.

Toxicity testing

Toxic effect of MOPBS chronic treatment on liver and kidney function test was determined by measuring creatinine, aspartate amino transferase (AST) and alanine amino transferase (ALT). All animals received a single dose of MOPBS 50 mg/kg, dexamethasone 5 mg/kg and vehicle control (normal saline with 2% DMSO) daily. At day 21, blood samples were collected from cardiac puncture. Total blood centrifuged at 2500 rpm for 10 min to separate serum. Serum samples were used for biochemical analysis (Khan et al. 2014).

Radiological and histological analysis

Radiological analysis of paw tissue was performed to investigate the effect of daily pretreatment of MOPBS 50 mg/kg on mice inflamed paw. At the end of experiments, mice were killed using chloroform. Right hind paw was removed and radiographed. Radiographs were evaluated for any sign of swelling of soft tissue, bone resorption and new bone formation by uing Philips 612 machine (40 kW for 0.01 s) (Arora et al. 2015; Cai et al. 2006). After X-ray analysis, inflamed paws were washed with saline and fixed with 10% formalin. After fixation, paw tissues were rinsed, dehydrated and embedded in paraffin. Paw tissue blocks were sectioned at 40 μm thickness. Haematoxylin and eosin staining was performed and observed by microscopy according to the method previously described (Cai et al. 2006; Khan et al. 2013a, b).

Inverted screen test and weight test

To assess the effect of MOPBS (50 mg/kg) acute and chronic treatment on muscle strength and motor function, Kodzeila’s screen test was performed at 0, 6 h, 6th and 21-day post-CFA administration. Each mouse was placed in the center of wire mesh screen and screen was inverted. Investigator was trained to hold the screen 40-50 cm above a smooth surface. Time taken by mice to hold the inverted screen was recorded using digital stop watch and score was assigned according to the protocol (Contet et al. 2001). The time at which the mouse fell was recorded to maximum of 60 s.

For full assessment of motor deficit, weight test was performed according to previously reported protocol (Deacon et al. 2002). Each weight was constructed using a thin wire mesh to which a length of steel chain consisting of several links from one to seven was attached. The first link weights 20 g and all others weight approximately 7 g. Therefore, the weights were 20, 27, 33, 46, 59, 72, 85 and 98 g. Each mouse was held by the tail and lowered in order to grasp the first weight (20 g). Time at which mouse successfully grasped the wire mesh, mouse was lifted up and stop watch was started. If the mice dropped the weight in less than 3 s, a second trial was made. A rest of 10 s was given to each animal between each lift. Score was assigned to each mouse.

Gastric toxicity test

The effect of MOPBS treatment was observed on gastric toxicity following P.O administration. The animals were divided into three groups (Vehicle Control received saline and 2% DMSO, piroxicam 10 mg/kg treated group and MOPBS 50 mg/kg treated group). Oral administration was continued for 3 days. At the end of day 3, animals from all groups were sacrificed. To investigate the possible gastric effect, histopathological analysis of stomach was performed as described somewhere else and observed for inflammatory cell infiltration and necrosis (Brzozowski et al. 2000).

Urinary and serum electrolyte measurement

Effect of MOPBS on Renal Na+ and K+ excretion rate was investigated. Animals were divided into three groups (MOPBS 50 mg/kg (i.p), Piroxicam 10 mg/kg (i.p) and vehicle control group (normal saline with 2% DMSO) (i.p). Each group contained five animals. Drug administration was continued for 3 days. For the last third day, animals were housed individually in metabolic cages and urine was collected over last 24 h period. Urine was stored at – 20 °C. At the end of experiments, animals from all groups were killed and blood was collected from cardiac puncture. Serum was separated by centrifugation and stored at – 80 °C (Terker et al. 2015). Na+ and K+ level was than determined in urine and serum sample.

Statistical analysis

Unless otherwise stated results are expressed as mean ± standard deviation (SD). The differences between the vehicle control and negative control group were tested with unpaired Student’s t test and one-way analysis of variance (ANOVA). A value of p < 0.05 was chosen as the criterion for statistical significance using Sigmaplot version 10.0, Chicago, USA.

Results

Effect of MOPBS on carrageenan-induced paw oedema, mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia

In first series of experiment, anti-inflammatory effect of MOPBS (1, 10, 50 or 100 mg/kg) was evaluated using carrageenan-induced acute inflammation model. MOPBS (1, 10, 50 or 100 mg/kg i.p) administration 1 h prior to carrageenan injection significantly reduced paw oedema at 4 h in dose-dependent manner as compared to carrageenan-induced group (Fig. 1b). The results obtained with vehicle control group and negative control (carrageenan-treated group) supported the results obtained for MOPBS treatment (Fig. 1b). As shown in Fig. 1b, the mice administered with higher concentration of MOPBS (50 and 100 mg/kg) showed a remarkable reduction in paw oedema as compared to lower concentration (MOPBS 1 or 10 mg/kg). It is noteworthy that the effect of MOPBS on paw oedema at higher concentrations (50 and 100 mg/kg) did not differ significantly. To confirm the inter-link between analgesic and anti-inflammatory action of MOPBS (1, 10, 50 or 100 mg/kg), anti-nociceptive properties of MOPBS were evaluated using Randall Selitto test, von Frey test and Hot plate test. MOPBS (50 and 100 mg/kg) i.p. administration showed a appreciable inhibition of carrageenan-induced mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia in mice after 4 h of carrageenan injection (Fig. 1c, d, e). Carrageenan-induced mechanical hyperalgesia and allodynia responses were significantly inhibited by dexamethasone (5 mg/kg).

On the basis of acute model results, it was observed that MOPBS has significant anti-inflammatory and anti-nociceptive properties at higher dose (50, 100 mg/kg) and there is no remarkable difference of effects between 50 and 100 mg/kg; therefore, it was selected for the next subsequent experiments.

Effect of MOPBS on paw oedema in CFA-induced chronic arthritis model in mice

Subplanter administration of CFA in mice right hind paw produced a significant chronic inflammation which was steadily maintained for 21 days. MOPBS 50 mg/kg significantly reduced paw oedema at 4 h (Fig. 2a) after CFA injection. Similarly, MOPBS significantly reduced paw oedema at days 14 and 21 (Fig. 2b). Dexamethasone, a positive control, also showed considerable inhibition of paw thickness throughout the arthritis model.

Fig. 2
figure 2

a Acute pretreatment of MOPBS (50 mg/kg) reduced CFA-induced paw oedema. Paw thickness was measured every 2 h after CFA administration from 0 to 6 h. b Effect of chronic pretreatment with MOPBS (50 mg/kg) on paw oedema. Paw thickness was measured after CFA injection from 0 to 21 days. The mRNA expression level of c TNF-α, d IL-1β, e IL-6 in paw tissues determined by quantitative real-time PCR. The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate significant differences from the CFA-treated group. (###) denotes comparison of vehicle control group (normal group) and CFA-induced group with MOPBS-treated group

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Effects of MOPBS on mRNA expression levels of cytokines in CFA-induced paw tissues

Quantitative RT-PCR results showed that CFA dramatically increased TNF-α, IL-1β and IL-6 mRNA expression levels in time-dependent manner (Fig. 2c, d, e). MOPBS pretreatment significantly reduced the mRNA expression level of TNF-α, IL-1β and IL-6 as shown in Fig. 2c, d, e.

Effect of MOPBS pretreatment on mechanical hyperalgesia and mechanical allodynia

Mechanical hyperalgesia was significantly inhibited by MOPBS (50 mg/kg) at 2, 4 and 6th h (p < 0.0001) (Fig. 3a). A significant decrease in mechanical nociception was also seen in CFA-induced group at day 1 and was reduced subsequently until day 21 (Fig. 3b). The i.p. administration of MOPBS (50 mg/kg) significantly inhibited mechanical allodynia in mice after 4 h of CFA injection as illustrated in Fig. 3c. CFA-induced mechanical allodynia was also remarkably inhibited by dexamethasone (5 mg/kg). When compared with negative control, MOPBS (50 mg/kg) showed a significant increase in paw withdrawal threshold at days 6, 14 and 21 (Fig. 3d).

Fig. 3
figure 3

a Acute pretreatment of MOPBS inhibited mechanical hyperalgesia. The paw withdrawal effect was recorded every 2 h after CFA administration from 0 to 6 h. b Chronic MOPBS pretreatment inhibited mechanical hyperalgesia. The paw withdrawal effect was measured after CFA injection from 0 to 21 days. c Effect of acute pretreatment of MOPBS on mechanical allodynia. The paw withdrawal effect was recorded every 2 h after CFA injection from 0 to 6 h. d Effect of chronic MOPBS pretreatment on mechanical allodynia. The paw withdrawal effect was recorded post-****CFA injection from 0 to 21 days. The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate notable differences from the CFA-treated group. (###) denotes comparison of vehicle control group (normal group) and CFA-induced group with MOPBS-treated group

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Effect of MOPBS on thermal hyperalgesia and cold allodynia

The i.pl administration of CFA into right hind paw significantly increased the thermal hyperalgesia (Fig. 4a). However, there was a marked reduction in thermal hyperalgesia after 2 and 4 h of CFA injection at day 1 in MOPBS-treated group (Fig. 4a). In chronic phase, a significant reduction of thermal pain was observed in MOPBS-treated group at days 14 and 21 (Fig. 4b). Cold allodynia was also investigated using cold acetone test. CFA-induced group showed an abnormal sensitivity to cold stimuli after 4 h of CFA injection. However, MOPBS pretreatment showed a significant reduction in cold pain after 4 h of CFA injection (Fig. 4c). Similarly, chronic MOPBS treatment significantly increased the threshold for cold pain after 1, 6 and 21 days of CFA injection (Fig. 4d).

Fig. 4
figure 4

a Effect of acute pretreatment of MOPBS on thermal hyperalgesia. The paw licking response was recorded every 2 h after CFA injection from 0 to 6 h. b Effect of chronic MOPBS pretreatment on thermal hyperalgesia. The paw licking response was measured post-CFA injection from 0 to 21 days. c Effect of acute pretreatment of MOPBS on cold allodynia. The paw withdrawal effect was recorded at 0 h and 4 h after CFA administration. d Effect of chronic MOPBS pretreatment on cold allodynia. The paw withdrawal effect was measured at day 1, 6 and 21 days after CFA administration. The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate notable differences from the CFA-treated group. (###) denotes comparison of vehicle control group (normal group) and CFA-induced group with MOPBS-treated group

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Effect of MOPBS on antioxidant enzymes in CFA-induced paw tissues and nitrite production in bold plasma

MOPBS treatment upregulated Nrf2 expression level (Fig. 5a). The mRNAs expression levels of HO-1, SOD2 were increased in MOPBS-treated group as compared to CFA-induced group (Fig. 5b, c). NO production was considerably high in CFA-treated group (Fig. 5d). However, daily treatment of MOPBS (50 mg/kg) significantly reduced NO concentration in blood plasma as shown in Fig. 5d. MOPBS significantly decreased the mRNA expression of VEGF (Fig. 6a).

Fig. 5
figure 5

The mRNA expression level of a Nrf2, b HO-1, c SOD2 determined by quantitative real-time PCR. d Chronic pretreatment with MOPBS reduced nitrite (NO) production in blood plasma. Animals were administered with MOPBS (50 mg/kg, i.p.), dexamethasone (5 mg/kg, i.p.), CFA and vehicle control. The NO concentration was determined using Griess reagent. The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate significant differences from the CFA-treated group. (###) denotes comparison of vehicle control group (normal group) and CFA-induced group with MOPBS-treated group

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Fig. 6
figure 6

a The mRNA expression level of VEGF determined by quantitative real-time PCR. b Soft tissue swelling in CFA-treated group as compared to MOPBS-treated group. Radiographic evidence of right hind paw from (1) vehicle control group, (2) CFA-induced group, (3) Dexamethasone-treated group and (4) MOPBS-treated group. c Histopathological analysis of tibio-tarsal joint from (1) vehicle control group, (2) CFA-induced group, (3) Dexamethasone-treated group and (4) MOPBS-treated group. Paw tissue blocks were sectioned at 4 μm thickness, stained with haematoxylin–eosin, and observed by microscopy (×40). Marked immune cell infiltration, synovial hyperplasia, damaged osteocytes, enlarged fibrils and cartilage destruction in CFA-treated group as compared to MOPBS-treated group. The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate significant differences from the CFA-treated group

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Effect of MOPBS on liver and kidney functions

To evaluate the possible toxic effects of MOPBS, kidney and liver enzyme levels were also investigated. Daily treatment of MOPBS (50 mg/kg) for 21 days did not produce any variation in normal liver and kidney function. Level of hepatic enzymes (ALT 96 ± 2.12 UI/L and AST 142 ± 1.41 UI/l and creatinine 0.5 ± 0.28 mg/dl) in MOPBS (50 mg/kg)-treated group did not show any significant alteration when compared with vehicle control group (ALT 94 ± 2.121 UI/l and AST 141 ± 1.41 UI/l and creatinine 0.4 ± 0.14 mg/dl) and dexamethasone (positive control) (ALT 97 ± 1.41 UI/l and AST 144.5 ± 2.12 UI/l and creatinine 0.35 ± 0.07 mg/dl).

Radiological studies and histological analysis

Soft tissue examination showed a remarkable reduction in swelling of right hind paw in MOPBS-treated group. There was predominant soft tissue swelling, bone resorption and cartilage erosion in CFA-induced group. Radiographs taken at 21 days of experiment showed a significant reduction in soft tissues swelling and marked cartilage repair in MOPBS-treated group (Fig. 6b). To explore further the infiltration of immune cells, HNE staining was performed. Histopathological analysis of tibio-tarsal joint of right hind paw showed a significant infiltration of immune cells, synovial hyperplasia, damaged osteocytes, cartilage and enlarged fibrils in CFA-treated group, while MOPBS-treated group showed repaired cartilage and just a few enlarged fibrils (Fig. 6c).

Effect of MOPBS pretreatment on motor co-ordination activity and muscle strength

Weight lifting and Kodzeila’s screen tests showed that MOPBS-treated group did not produce any abnormal effect on motor function. There was no significant impairment in motor co-ordination or muscle strength as well (Fig. 7a, b).

Fig. 7
figure 7

a Effect of MOPBS on muscle strength and motor co-ordination of animals was measured using inverted tray method at 0, 6th h, 6th and 21st day as described in materials and method section. b Effect of MOPBS treatment on muscle strength and motor function of animals was measured using weight test at 0, 6th h, 6th and 21st day. The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate significant differences from the CFA-treated group. (###) denotes comparison of vehicle control group (normal group) and CFA-induced group with MOPBS-treated group

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Effect of MOPBS treatment on gastric mucosa

Haematoxylin–eosin staining of stomach observed by microscopy (×40) indicated that there are no necrosis, immune cell infiltration and hemorrhagic lesions in MOPBS-treated group. Piroxicam-treated group showed immune cell infiltration (Fig. 8).

Fig. 8
figure 8

Haematoxylin–eosin staining of stomach observed by microscopy (×40).Gastric effect of daily treatment with MOPBS 50 mg/kg (P.O). The data are expressed as the mean ± SD (*) p < 0.01, (**) p < 0.001 and (***) p < 0.0001 indicate significant differences from the CFA-treated group

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Effect of MOPBS treatment on urinary and serum electrolytes (Na+ and K+) excretion rate

MOPBS treatment did not alter serum and urinary Na+ and K+ level when compared with vehicle control-treated group (Table 1).

Table 1 Urinary and blood electrolyte levels
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Discussion

Carrageenan-induced paw oedema and CFA-induced arthritis are well-known inflammatory models and have been extensively used for many years for evaluation of anti-inflammatory and analgesic activity of various new therapeutic agents (Costa et al. 2004; Walz et al. 1971). CFA-induced arthritis mice model was developed in the present study to determine the effect of MOPBS on acute paw oedema after CFA administration at day 1 as previously determined by Khan et al. (2013a, b). CFA-induced chronic arthritis mice model has been previously developed in various research studies for evaluation of chronic inflammation and hyperalgesia (Horváth et al. 2016; da Silva et al. 2016). CFA-induced chronic inflammation consists of primary phase and secondary chronic phase. Primary phase is inflammatory phase associated with generation of pro-inflammatory mediators such as TNF-α, IL-1β and IL-6 and secondary phase involves generation of autoantibodies (Walz et al. 1971). Chronic arthritis is reflected by significant increase in paw thickness after subplanter injection of CFA (Yu et al. 2006). MOPBS pretreatment has significantly reduced paw oedema. CFA-induced arthritis has been widely used as a laboratory model for evaluation of chronic pain (Bertorelli et al. 1999). Arthritis pain is spontaneous pain results from sensitization of nociceptive system. Inflammation and pain in joint are interlinked. In inflamed joints, afferent neurons are activated and pressure on inflamed joint activates A-δ fibers and C fibers which in turn releases several neuropeptide (such as substance P and glutamate). Activated sensory fiber then stimulates articular nociceptors and, thus, results in persistent joint pain (SCHAIBLE et al. 2002). MOPBS pretreatment remarkably inhibited mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia during the entire course of study.

Pharmacologically, upregulation of several pro-inflammatory mediators and cytokines such as NO, TNF-α, IL-1β and IL-6 produces a robust inflammatory response (Feldman and Maini 2008). TNF-α is an autocrine stimulator and a potent paracrine inducer, therefore, exaggerates the production of otheSr pro-inflammatory mediators such as NO, IL-1β and IL-6 (Feldman and Maini 2008). In vitro studies indicated that IL-1β potentially increases the metalloproteinases generation and osteoclast activation (Barksby et al. 2007). TNF-α is known to be a systemic marker of inflammation and plays a significant role in cartilage damage and bone degradation (MacNaul et al. 1990). Therefore, inhibition of production and release of inflammatory mediators is thought to be effective in treating chronic inflammatory diseases such as RA. In support to this, MOPBS pretreatment not only decreased the production of NO but qRT-PCR analysis also showed that the MOPBS significantly reduced the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6).

In inflammatory diseases, an excessive release of reactive oxygen species (ROS) has been observed due to the over-activation of phagocytes and neutrophils (Hitchon and El-Gabalawy 2004). Increase ROS generation in synovial membrane results in oxidative burst. Thus, it damage lipids, protein, nucleus acid, collagen and worsens the disease condition. In the current study, the intraplanter administration of CFA results in reduction of endogenous antioxidants such as SOD2 (superoxide dismutase) and heme oxygenase (HO-1) and increased pro-oxidant level (NO) in CFA-treated group (negative control) as compared to vehicle control group. Dexamethasone significantly enhanced the expression of Nrf2 by 1500% which is comparable with expression of Nrf2 induced by MOPBS. Nrf2 transcription plays a significant role in conferring cytoprotection against oxidative stress. Nrf2 exerts its biological regulatory effect by controlling the expression of genes required for free radical scavenging, detoxification of xenobiotics and maintenance of the redox potential. Nrf2 regulated genes, known as phase II enzymes such as SOD2, HO-1 (Talalay et al. 2003). MOPBS pretreatment significantly increase the mRNA expression level of Nrf2, SOD2 and HO-1.

VEGF promotes angiogenesis and pannus formation and is also responsible for recruitments of immune cells towards inflammatory sites due to the increasing vascular permeability (Zuo et al. 2014). Several in vivo studies indicated that VEGF produces the characteristic features of arthritis by stimulating the chondrocytes to express metalloproteinases via interaction with VEGF receptors (Lu et al. 2000). Increased NO production has been reported in VEGF-stimulated chondrocytes that are the direct inducer of chondrocytes apoptosis (Zuo et al. 2014). VEGF stimulation of synovial chondrocytes leads to expression of several pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. Blockage of VEGF signaling results in reduction of arthritis severity (Lu et al. 2000). Therefore, it was investigated that MOPBS reduced the mRNA expression level of VEGF as compared to CFA-treated group. In the present study, MOPBS not only protected synovial membrane and repaired cartilage damage but also improved health status through its antioxidant properties. Furthermore, MOPBS also improved clinical signs of RA disease such as paw oedema and arthritis pain and showed no toxic effect on gastric mucosa, renal function, hepatic function and motor co-ordination. Conclusively, the current study provides several evidences that MOPBS, a synthetic derivative of benzenesulphonamide, produced significant anti-inflammatory and analgesic activities.