Exploring the possible mechanism involved in the anti-nociceptive effect of β-sitosterol: modulation of oxidative stress, nitric oxide and IL-6

Β-sitosterol is a phytosterol, documented to possess various activities including protection against inflammation, diabetes and Alzheimer’s disease. The current investigation was designed to explore the analgesic potential of β-sitosterol and the possible molecular mechanism involved in the observed effect. β-sitosterol was administered at varying doses of 10, 20, and 40 mg/kg before subjecting the mice to acetic acid and formalin challenges. The number of writhings in acetic acid and the number of flinchings and foot tappings were quantified in the formalin test. For mechanistic studies, substance P (cyclooxygenase-2 (COX-2) stimulator) and L-Nitro arginine methyl ester (L-NAME) (nitric oxide synthetases (NOS) inhibitor) and L-arginine (nitric oxide precursor) were administered before β-sitosterol treatment. β-sitosterol (10, 20, 40 mg/kg) treatment significantly reduced acetic acid-induced writhings and ameliorated the formalin-induced inflammatory phase dose-dependently. Whereas, 40 mg/kg dose of β-sitosterol abrogated the formalin-induced neurogenic phase. Substance-P abrogated the effect of β-sitosterol in both neurogenic and inflammatory phases. Whereas, L-arginine only abrogated the inflammatory phase. In biochemical analysis, β-sitosterol treatment reduced the level of interleukin-6 (IL-6), thiobarbituric acid reactive substances (TBARS) and increased the level of reduced glutathione (GSH). Furthermore, L-arginine and substance-P abrogated the GSH increasing and TBARS lowering effect of β-sitosterol (40 mg/kg). Overall, the current study delineated that β-sitosterol may induce an anti-nociceptive effect via inhibiting the IL-6, oxidative stress, cyclo-oxygenase and nitric oxide.


Introduction
Pain can be defined as a distressing sensation that may be localized or widespread with an emotional experience linked to tissue damage. Pharmacoepidemiologic data has revealed that about 28 million adults in the United Kingdom suffer from chronic pain at some time in their life and 1 out of 5 adults in the United States had reported chronic pain (Stevens and Stephens 2018;Kuehn 2018). Chronic pain is often an indicator of serious problems such as fibromyalgia, peripheral neuropathy, arthritis and malignancy (Yam et al. 2018). Pain is a serious problem that contributes to the poor quality of life and increases the cost of healthcare in society (Singh et al. 2018a). The activation of pain receptors in primary afferent nerve fibers, which include the myelinated Aσ-fiber and unmyelinated C-fibers, is linked to the perception of pain. During homeostasis, when there is no noxious stimulus present, both nociceptors are inactive and as soon as there is exposure to a noxious stimulus, these are activated (Yam et al. 2018). Various mediators such as prostaglandins, nitric oxide (NO), amino acid neurotransmitters and pro-inflammatory cytokines are involved in pain sensation (Singh et al. 2018b). Routine clinical management of pain and inflammation involves the use of non-steroidal antiinflammatory drugs (NSAIDs), opioids and corticosteroids whereas neuropathic pain is managed with the help of anticonvulsants such as gabapentin (Moore and Gaines 2019). However, these agents have limited utility because of the adverse effects caused during their use to treat pain (Brusco et al. 2017). Various side effects caused by the currently employed drugs such as ulcerogenic and renal damage with NSAIDS, constipation, addiction, physical dependence and 1 3 respiratory depression with opioids, and behavioral problems with gabapentin, result in poor patient acceptability (Singh et al. 2017). Due to the aforementioned limiting factors, there is increased impetus on new research with an aim to develop safe and more effective pain treatments.
Over the years, a lot of work has been put forward to explore natural products for the management of pain and co-morbid conditions (Chakraborty and Majumdar 2020;Jahromi et al. 2021;Brusco et al. 2017). Recently, phytosterols have been explored in various ailments. Phytosterols are a group of compounds that are found in plants chiefly as components of the plant membranes (Lesma et al. 2018). They play an important role in cellular differentiation, and proliferation and also possess cholesterol-lowering potential (Lesma et al. 2018;Jones and AbuMweis 2009). Major phytosterols studied for their pharmacological significance include β-sitosterol, stigmasterol, campesterol, stigmastanol and campestanol. β-sitosterol is a dietary phytosterol found mainly in saw palmetto, pumpkin seeds, black cumin seeds, pecans, cashew fruits, avocados, rice bran, wheat germ, corn oil, soybean and sea-buckthorn (Moore and Gaines 2019; Moreau et al. 2002). β-sitosterol has been documented to be a safe and effective nutritional supplement (Gupta 2020;Paniagua-Pérez et al. 2005). It possesses various pharmacological activities such as anti-proliferative (Cilla et al. 2015), anti-cholesterolemic (Shefer et al. 1988), anti-colitis effect (Lee et al. 2012), anti-alzheimer anti-oxidant and anti-diabetic effect (Ayaz et al. 2017) and protective effect in benign prostate hyperplasia (Wilt et al. 2001).
However, there are no studies indicating the effect of β-sitosterol in neurogenic and inflammatory hyperalgesia and possible mechanism leading to the anti-inflammatory and anti-nociceptive effect of β-sitosterol. Therefore, the present study was designed to investigate the antinociceptive and anti-inflammatory effect of β-sitosterol by employing acetic acid and formalin tests, along with possible mechanisms involved in mediating the analgesic effect.

Materials and methods
Chemicals Β-sitosterol was purchased from Himedia Laboratories (India). Indomethacin, Substance P, L-arginine and L-NG-Nitro arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich (USA). Interleukin-6 (IL-6) ELISA kit was purchased from Krishgen Biotech (India). All the other reagents and chemicals of analytical grade were purchased from authorized dealers (Vinny Scientific Store, Amritsar, Punjab, India). β-sitosterol was suspended in carboxymethyl cellulose by triturating thoroughly followed by micronizing in an ultrasonicator. The suspension was freshly prepared before the experiments.

Animals
Swiss albino mice of either sex with body weight in a range of 20-25 g (10 weeks age) were used. Animals were procured from the National Institute of Pharmaceutical Education and Research, Mohali, India and kept in the central animal house of Guru Nanak Dev University, maintained under standard environmental conditions 25 ± 5 °C with 12-h light/ dark cycle and water and feed were provided ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Guru Nanak Dev University, Amritsar (226/CPCSEA/2018/41). All experimental procedures were undertaken as per the guidelines for the use and care of laboratory animals.

Experimental protocol
Formalin-induced nociception and acetic acid-induced writhing tests were used to study the analgesic potential of β-sitosterol. β-sitosterol (10, 20 and 40 mg/kg) and standard drug indomethacin (10 mg/kg) which were administered intraperitoneally, prior to acetic acid and formalin treatment in respective tests. For mechanistic studies, the role of nitric oxide (NO) was studied by using modulator challenge with NO precursor L-arginine (40 mg/kg; ip) and nitric oxide synthase (NOS) blocker, L-NAME (10 mg/kg, ip), whereas the role of COX (cyclooxygenase) was studied by using substance P (10 µg/kg; ip), a neurokinin known to stimulate COX-2 (Castellani et al. 2009;Sio et al. 2010;Koon et al. 2006). L-NAME, L-arginine and substance were administered 30 min prior to the β-sitosterol (40 mg/kg, ip). After β-sitosterol treatment, 2% formalin was injected into the hind paw and the licking and tapping behavior was recorded for 60 min. After behavioral evaluation in the formalin test, the animals were anesthetized (ketamine 50 mg/kg) and blood samples were taken for serum IL-6 estimation. Thereafter, animals were killed and the brain samples were extracted for oxidative stress analysis. The formalin-injected paw was excised for the histological examination.

Acetic acid-induced writhing test
The acetic acid-induced writhing test was used to evaluate the peripheral antinociceptive activity. Acetic acid-induced writhings were induced by injecting acetic acid (0.6% v/v) intraperitoneally at a dose of 10 ml/kg. The number of writhings was counted for a period of 30 min post administering of acetic acid as an indicator of hyperalgesia. The test and standard drugs were administered 30 min before the acetic acid treatment (Owoyele et al. 2010).

Formalin-induced licking and tapping test
In this method, 2% formalin at a dose of 50 µL was injected subcutaneously into the intraplantar region of the right hind paw and the number of licking and tappings was counted for a period of 60 min as a measure of hyperalgesia. The first phase of 0-5 min is the neurogenic phase and the second phase of 20-25 min is the inflammatory phase. The test and standard drugs were administered 30 min before formalin injection (Lucetti et al. 2010;Ghorbanzadeh et al. 2016).

Serum IL-6 estimation
The animals were anesthetized with ketamine (50 mg/kg) after formalin test. The blood samples were collected by the retro-orbital route. The serum was separated from the blood by spinning the blood samples at 3000 g for 10 min. Serum IL-6 estimation was done by using commercially available ELISA kits (Krishgen Biotech).

Preparation of brain homogenate
Brain homogenate was prepared by adding the dithiothreitol (15.4 mg) and triton X (500 mg) to the phosphate-buffered saline solution of pH 7.4. Homogenate was spun at 2000 g for 10 min to obtain a clear supernatant. The pellets were discarded and the supernatant was used for the estimation of reduced glutathione and lipid peroxidation.

Thiobarbituric acid reactive substances (TBARS) estimation
An increased level of TBARS in tissue is considered an index of increased lipid peroxidation in tissue. 0.4 ml of TCA-TBA-HCl reagent was taken and added to 0.2 ml of supernatant. The solution was kept in a boiling water bath for 5 min, brought to room temperature and spun at 10,000 g for 10 min. The colored supernatant was collected and measured spectrophotometrically at 535 nm (Kumar et al. 2010).

Reduced glutathione (GSH) estimation
Ellman method was used for measuring the glutathione content in brain tissue. 1 ml of 0.3 M disodium hydrogen phosphate was added to the 0.25 ml of supernatant followed by the addition of 0.125 ml of 0.001 M freshly prepared DTNB.
The resultant yellow-colored solution was measured spectrophotometrically at 412 nm (Kumar et al. 2010).

Haematoxylin and eosin (H & E) staining of the treated right hind paw
The histological analysis of paw tissue was carried out to investigate the effect of β-sitosterol on formalin-induced paw inflammation. Skin from the paw was isolated and preserved in 10% formalin solution for 24 h (Khan et al. 2018). The tissue was dehydrated by exposure to alcohol, immersed in xylene and then embedded in paraffin (Park et al. 2013). Sections of 3-4 µm thickness were cut and placed on a slide using the mounting medium. After mounting the section, paraffin wax was removed by gently warming the slide followed by washing with xylene. This was followed by alternate washings with varying concentrations of alcohol (70%, 80%, 90% and 100%) and water to prevent dehydration. Thereafter, the sections were stained with haematoxylin for 15 min. The washing of stained sections was done with water. Then the sections were treated with a 1% acid-alcohol mixture for 20 s. The acid alcohol mixture was washed off with water and counterstained with a 1% aqueous solution of eosin for 2 min. The excess eosin was washed with water and sections were dehydrated with alcohol. The dehydrated sections were mounted with Canada balsam as a mounting agent under a coverslip carefully to avoid bubbles. The slides were observed under the microscope at 40x (Singh 2018b).

Statistical analysis
The data were expressed as mean ± standard error mean (SEM). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by post-hoc analysis by Tukey's multiple comparison test using Graph pad Prism software version 7.0. p < 0.05 was considered significant.

Effect of β-sitosterol on acetic acid-induced writhings
Intraperitoneal injection of 0.6% acetic acid induced writhing response in mice. Pretreatment with β-sitosterol significantly reduced the number of writhings in comparison to the acetic acid controls in a dose-dependent manner (Fig. 1A). β-sitosterol at three different doses of 10, 20 and 40 mg/kg reduced the acetic acid-induced writhings by 26.51%, 52.27% and 66.66%, respectively, as compared to the acetic acid control mice. Pretreatment with the standard drug indomethacin (10 mg/kg, i.p) significantly reduced the number of writhings by 53.03% (percentage antinociceptive activity) (Fig. 1B).

Effect of β-sitosterol treatment on formalin-induced licking and tappings
Intraplantar injection of 2% formalin induced licking and foot tapping behavior in mice. The licking and tapping behavior in the formalin test was plotted as the area under the curve (AUC) for a period of 60 min. It has been observed that administration of formalin produced a biphasic pain response with the first phase (neurogenic hyperalgesia) evident from 0 to 5 min and the second phase (inflammatory hyperalgesia) evident from 20 to 25 min after the formalin challenge. Pretreatment with β-sitosterol at doses of 40 mg/kg reduced the number of licking and tappings by 19.92%, in the first neurogenic phase (0-5 min) as compared to the formalin control group. However, no significant change was observed with 10 and 20 mg/kg of β-sitosterol in the first neurogenic phase as compared to the formalin control group, Whereas in the inflammatory phase (20-30 min), 20 and 40 mg/kg β-sitosterol treatment significantly reduced the number of licking and tappings by 32.49% and 86.50% respectively, as compared to the formalin control group. However, no significant change was observed with 10 mg/ kg of β-sitosterol in the inflammatory phase as compared to the formalin control group. Moreover, indomethacin (10 mg/kg, ip) treatment attenuated the inflammatory phase, whereas no significant change was observed in the neurogenic phase ( Fig. 2A, B).

Effect of substance P on the observed analgesic action of β-sitosterol
Pretreatment of mice with Substance P (COX-2 stimulator) at a dose of 10 µg/kg significantly abrogated the effect of β-sitosterol (40 mg/kg) in both neurogenic and inflammatory phases. A significant increase in licking and tapping of the paw was observed in the Substance P pretreated group as compared to the β-sitosterol (40 mg/kg) treated group (Fig. 3).

Effect of L-arginine and L-NAME on the observed analgesic action of β-sitosterol
Pre-treatment with L-arginine (NO precursor) significantly abrogated the effect of β-sitosterol (40 mg/kg) in the inflammatory phase. No significant change was observed in the neurogenic phase. A significant increase in licking and tapping of the paw was observed in the L-arginine pretreated group as compared to the β-sitosterol (40 mg/kg) treated group, whereas pre-treatment with L-NAME (NOS inhibitor) did not alter the effect of β-sitosterol in any phase of formalin-induced nociception (Fig. 4).

Serum IL-6 estimations
Formalin-administered mice showed a significant increase in plasma levels of IL-6 as compared to the normal untreated group. β-sitosterol (40 mg/kg) treatment significantly reduced the levels of IL-6 by 39.09% as compared to the formalin control group (Fig. 5).

TBARS
Brain tissue of formalin-treated mice showed a significant increase in the TBARS level as compared to the untreated normal control group. β-sitosterol (10, 20 and 40 mg/kg) and indomethacin (10 mg/kg) significantly reduced the level of TBARS in the brain as compared to that of the formalin control group. The maximum effect of β-sitosterol was observed at 40 mg/kg. In mechanistic studies, L-arginine and substance-P pretreatment significantly abrogated the TBARS-lowering effect of β-sitosterol (40 mg/kg). A significant increase in TBARS level was observed in the L-arginine and the substance-P pretreated group as compared to the β-sitosterol treated group. However, L-NAME pretreatment did not alter TBARS lowering effect of β-sitosterol (Fig. 6).

Reduced glutathione content
Brain tissue of formalin-treated mice showed a significant decrease in the GSH level as compared to the untreated normal control group. β-sitosterol (20 and 40 mg/kg) and indomethacin (10 mg/kg) significantly increased the level of GSH in the brain as compared to that of the formalin control group, whereas β-sitosterol (10 mg/kg) treatment had no significant effect on brain GSH level as compared to that of the formalin control group. The maximum effect of β-sitosterol was observed at 40 mg/ kg. In mechanistic studies, L-arginine and substance-P Fig. 2 A Effect of β-sitosterol (10, 20 and 40 mg/kg) on formalin-induced licking and tapings; and B effect of β-sitosterol (10, 20 and 40 mg/ kg) on formalin-induced licking and tapings in both neurogenic and inflammatory phase. Data were expressed as mean ± SEM. Statistical differences were determined by using one-way ANOVA followed by Tukey's multiple comparison test. a p < 0.05 vs. formalin control, b p < 0.05 vs. indomethacin, c p < 0.05 vs. β-sitosterol 10 mg/ kg, d p < 0.05 vs. β-sitosterol 20 mg/kg pretreatment significantly abrogated the brain GSH increasing the effect of β-sitosterol (40 mg/kg). A significant decrease in brain GSH level was observed in the L-arginine, L-NAME and substance-P pretreated group as compared to the β-sitosterol treated group (Fig. 7).

H & E staining of paw
Microscopic observation on histological examination of paw skin indicated an increase in the number of inflammatory cells in formalin-exposed paw skin as compared to untreated paw skin. Administration of β-sitosterol (40 mg/kg) reduced the number of inflammatory cells as compared to formalinexposed skin. The effect was comparable to the standard drug indomethacin. L-arginine and substance P treatment significantly alleviated the protective effect of β-sitosterol on paw skin as evidenced by an increased number of inflammatory cells as compared to the β-sitosterol treated group (Fig. 8).

Discussion
In the current investigation, the analgesic and anti-inflammatory activity of β-sitosterol and the possible molecular pathway involved in the observed effect have been studied. For pain assessment, two animal models, namely acetic acid-induced writhing and formalin-induced nociception in mice were used. Intraperitoneal injection of 0.6% acetic acid is documented to cause contraction of abdominal muscles accompanying the extension of hind legs and trunk twist, termed as writhing and taken as an indicator of pain (Feng et al. 2003;Franca et al. 2001). Various inflammatory mediators such as prostaglandin, bradykinin and proinflammatory cytokines such as Interleukin-1β (IL-1β) and IL-6 are documented to be released due to activation of chemo-sensitive nociceptors after acetic acid injection which is responsible for the induction of writhings in mice (Pigatto Fig . 3 Effect of substance P on antinociceptive effect of β-sitosterol (40 mg/kg) in formalin-induced nociception test. Data were expressed in mean ± SEM. Statistical differences were determined by using twoway ANOVA followed by Tukey's multiple comparison test. a p < 0.05 vs. formalin control, b p < 0.05 vs. β-sitosterol Fig. 4 Effect of L-arginine and L-NAME on antinociceptive effect of β-sitosterol (40 mg/kg) in formalin-induced nociception. Data were expressed in mean ± SEM. Statistical differences were determined by using one-way ANOVA and Tukey's test. a p < 0.05 vs. formalin control, b p < 0.05 vs. β-sitosterol  Ikeda et al. 2001;Nirmal 2011). Formalin injection into the intraplantar region of the hind paw of mice shows biphasic responses of pain: neurogenic phase (early phase) and inflammatory phase (late phase) due to activation of C-fibers in the form of foot tapping and licking (Du et al. 2007;Cho et al. 2006). The early phase appears immediately after injection and lasts for 5 min due to the direct effect of formalin on the nociceptors and primary afferent nociceptor activity detected by Aδ-fibers mainly mediated by neurokinins (Cho et al. 2006), whereas the late phase lasts for 20-30 min and arises from nociceptive spinal neuronal hyperactivity mediated by prostaglandins, bradykinin, and nitric oxide (Demsie et al. 2019;Adedayo et al. 2019). In the present investigation, β-sitosterol treatment significantly abrogated acetic acid-induced hyperalgesia in a dosedependent manner. In the formalin-induced nociception test Fig. 6 Effect of various interventions on brain TBARS level in formalin treated mice. Data were expressed in mean ± SEM. Statistical differences were determined by using one-way ANOVA and Tukey's test. a p < 0.05 vs. normal control, b p < 0.05 vs. formalin control, c p < 0.05 vs. indomethacin, d p < 0.05 vs. β-sitosterol 10 mg/ kg, e p < 0.05 vs. β-sitosterol 20 mg/kg, f p < 0.05 vs. βsitosterol 40 mg/kg. A 0.1% of carboxymethyl cellulose (CMC) was used as a vehicle Fig. 7 Effects of various interventions on brain GSH level in formalin-treated mice. Data were expressed in mean ± SEM. Statistical differences were determined by using one-way ANOVA and Tukey's test. Pain and inflammation are pathological conditions that are mediated through complex pathways. Various studies show the involvement of NO and COX pathways in pain and inflammation. COXs are well-established mediators in the development of inflammatory disease (Chen et al. 2013). Out of the two isoforms of COX, COX-1 works as a housekeeping constitutive enzyme and COX-2 is activated during inflammatory conditions (Singh et al. 2018a). COX-1 and COX-2 release prostaglandins that are involved in regulating body temperature, prostacyclins for the smooth working of the cardiovascular system and prostanoids in immune system maintenance (Kaur et al. 2019). During inflammatory conditions, the enzyme COX-2 generates prostaglandins E2 which further causes various inflammatory diseases (Norregaard et al. 2015). Besides this, nitric oxide is an intracellular messenger that is released locally by inflammatory stimuli and has been involved in peripheral pain perception and inflammation (Tripathi et al. 2007). NO is a free radical, synthesized by NOS-catalyzed conversion of L-arginine to L-citrulline in presence of oxygen and other cofactors (Freire 2009). Studies also indicate an important role of NO in the production of acute and chronic pain at both central and peripheral sites (Cury et al. 2011). The excessive production of NO enhances the release of reactive oxygen species (ROS) including malondialdehyde, superoxide, peroxynitrite, etc. (Singh et al. 2018b). Studies have revealed that increased production of NO can activate COX which increases the production of inflammatory endopero-oxides and prostaglandins (Posadas et al. 2000). In the current study, substance-P pretreatment abrogated the effect of β-sitosterol in both neurogenic and inflammatory phases of formalin-induced nociception, whereas L-arginine abrogated the effect of β-sitosterol only in the inflammatory phase β-sitosterol (40 mg/kg). Moreover, no change was observed with L-NAME pretreatment, thus delineating that β-sitosterol might exert its analgesic effect by inhibiting NO and COX-2.
Oxidative stress occurs due to an imbalance between antioxidants and oxidant markers. Oxidative stress plays a crucial role in the development and maintenance of inflammation. Oxidative insult-induced inflammatory stress leads to various chronic diseases (Lugrin et al. 2014; Piao et al. Arrows in the figure indicate the infiltration of inflammatory cells. A 0.1% of carboxymethyl cellulose (CMC) was used as a vehicle 2021). Oxidative stress plays an important role in the maintenance of pain and pathogenesis of the inflammatory process by encouraging the production of various pro-inflammatory cytokines such as IL6, tumor necrosis factor-α (TNF-α), etc. through the activation of neutrophils (Santiago et al. 2015;Kiasalari et al. 2017). The previous reports have documented that the administration of formalin in a rat's hind paw upregulates NOS expression and increased serum NO level (Lam et al. 1996). In this study β-sitosterol significantly alleviated the formalin-induced increased oxidative stress as evidenced by a reduced level of TBARS and an increased level of GSH. Pretreatment with L-arginine and substance-P significantly abrogated the GSH-enhancing and TBARS-lowering effect of β-sitosterol (40 mg/kg). Moreover, the β-sitosterol treatment also reduced the formalin-induced increased level of IL-6. These findings delineate the oxidative and inflammatory stress-alleviating potential of β-sitosterol.
In conclusion, the results of the current investigation provide evidence that β-sitosterol has an appreciable analgesic activity. The observed analgesic activity of β-sitosterol involves a complex intermediation of cyclooxygenase and nitric oxide inhibition in addition to its anti-oxidative activity.
Author contributions KK, LS, AK performed all the experiments and helped in writing the manuscript. RB conceptualized the study.

Funding Nil.
Data availability All data generated or analyzed during this study is included in this article.
Code availability Not applicable.

Conflict of interest
The authors declare no conflicts of interest.

Ethical approval
The entire study involving the use of mice was approved by the Institutional Animal Ethics Committee (Approval No. (226/CPCSEA/2018/41) and the experiments were conducted according to ethical guidelines of the Ministry of Environment and Forests, Government of India.

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