Discussion
Effect of atorvastatin or glucosamine sulfate on osteoarthritic rats :
The histopathological examination of non-treated OA rats showed several osteoarthritic changes that are following Bagi et al., 2015; Marino-Martinez et al., 2019. Surgically-induced models of OA largely mimic the human OA histopathologically (Kuyinu et al., 2016).
Glucosamine treated group showed improvement of histopathological changes that occurred in the non-treated OA group in agreement with Wen et al., 2010 and Waly et al., 2017. Atorvastatin treated group showed a significant improvement of the histopathological score with an improved microscopic picture of the examined knee joint specimens to be almost near normal. These results are in agreement with Pathak, Lingaraju, et al., 2015 and Gaballah et al., 2015.
A significant increase in the maximum angle of knee extension in the osteoarthritic group, as a sign of joint stiffness, is in agreement with previous studies (Choi et al., 2015; Phan et al., 2015). The joint stiffness that occurs in OA mainly results from a chronic inflammatory process that progresses and leads to fibrosis and then dysfunction of the joint (Kim et al., 2012). Glucosamine and atorvastatin treated groups showed significant correction of the joint stiffness found in the non-treated osteoarthritic group. This improvement of the joint stiffness is assumed to be due to their anti-inflammatory effect that reducing the joint fibrosis.
In the present study, surgical induction of OA was associated with a significant increase in IL-1 β in agreement with previous studies by (Wei et al., 2018; Castrogiovanni et al., 2019). IL-1β alters the homeostatic balance of chondrocytes as it suppresses anabolic activity, and it decreases the expression of type II collagen and aggrecan and it inhibits glucuronosyltransferase which plays an important role in glycosaminoglycan biosynthesis. Also, it stimulates articular cartilage breakdown by increasing the expression of MMPs, chondrocyte apoptosis, and increasing the production of inflammatory mediators and reactive oxygen species (ROS)(Kapoor et al., 2011; Mobasheri and Batt, 2016).
The increase in IL-1β in OA rats was associated with a significant increase in serum level of MMP-13 following Ali et al., 2017; Castrogiovanni et al., 2019. Under normal conditions, there is a dynamic equilibrium between the synthesis and the degradation of the extracellular matrix component in osteoarthritic states, a disruption of matrix equilibrium leads to apoptosis of chondrocytes and cartilage degradation (Maldonado and Nam, 2013).  MMPs are the most important proteinases responsible for extracellular matrix degradation. MMPs are activated by abnormal environmental insults, including high-magnitude mechanical stress, inflammatory mediators (Shiomi et al., 2010).
The increase of IL-1β following the induction of OA was significantly reduced by glucosamine. This finding was in line with Shahine & Elhadidi, 2014. Glucosamine is assumed to decrease the serum level of IL-1β as it decreases interleukin 1-induced gene expression (Rovati et al., 2012). Glucosamine also limited the rise of MMP-13 produced by the induction of OA. This effect is in agreement with studies of (Rovati et al., 2012; Gibson et al., 2014).
Atorvastatin treated group in this study showed a significant reduction in the rise of IL-1 β in agreement with Barsante et al., 2005; Simopoulou et al., 2010. This decrease in IL-1 β level with atorvastatin is due to inhibition of production of isoprenoid derivatives that results in inhibition of NF-κB that regulates transcription of many inflammatory mediators including the IL-1 β and mutagenic signaling pathway (Baker et al., 2011).
Administration of atorvastatin to OA rats showed a significant reduction in the rise of MMP-13. Simopoulou et al., 2010 suggested that atorvastatin may have possible chondroprotective effects, mainly by decreasing cartilage degradation protein MMP13. Pathak et al., 2015b noticed that atorvastatin significantly inhibited the IL-1β-induced increased production of MMP-13 in an in-vitro OA model.
The effect of statins on MMPs may be due to inhibition of mevalonate synthesis which in turn plays an essential role in the regulation of several cellular mechanisms, including cytoskeletal dynamics and endocytic/exocytic transport. They are involved in MMP secretion, as well as transcription and synthesis of inflammatory cytokines and reactive oxygen species (Kavalipati et al., 2015).
Levels of reduced GSH in the erythrocyte lysate of osteoarthritic rats were reduced suggesting increased oxidative stress following Cifuentes et al., 2010; Regan et al., 2008. Increased ROS in OA is mainly associated with the reduction of the cartilage mass due to both inhibiting synthesis of cartilage matrix and inducing cartilage matrix breakdown (Henrotin et al., 2005). The glucosamine treated group in this study showed a significant elevation of the reduced GSH. Glucosamine was shown to possess antioxidant capacity (Katoh et al., 2017; Dai et al., 2018).
Atorvastatin treated group in this study showed a significant elevation of the decreased GSH. A similar antioxidant effect of atorvastatin has been reported by Pathak, Balaganur, et al., 2015 and Gaballah et al., 2015, who noticed that the GSH levels were restored to the normal by atorvastatin in an experimental model of osteoarthritis. The antioxidant effect of atorvastatin could be due to either increased biosynthesis of GSH or reduced oxidative stress. A recent study by (Hosseinzadeh et al., 2019) declared that atorvastatin enhanced the mRNA expression of antioxidant enzymes including glutathione peroxidase in cultured chondrocytes. Atorvastatin mitigated the production of ROS by endothelial cells by inhibiting NADPH oxidase activity via Rho-dependent mechanisms. Moreover, atorvastatin binds to erythrocyte membrane phospholipids or to lipoprotein fractions to prevent the diffusion of free radicals generated under oxidative stress (Bellosta et al., 2000).
Direct assessment of the anti-inflammatory and analgesic effects: It was found that sub-plantar injection of carrageenan produced a significant increase in paw thickness measured after 1, 2, 3, 4, 24h. These results came in agreement with Sadeghi et al., 2014 and Antonisamy et al., 2017. The carrageenan-treated group also showed a significant decrease in the nociceptive threshold that was parallel with Kuedo et al., 2016; Marius et al., 2018. The indomethacin treated group showed a significant reduction of the increased paw thickness produced by carrageenan in agreement with Uzkeser et al., 2012 and Okhuarobo and Ozolua, 2017. It also showed a significant analgesic effect in agreement with Kuedo et al., 2016; Uzkeser et al., 2012. Indomethacin was used in the present study as a well-known analgesic anti-inflammatory standard drug to which the tested drugs are compared.
Glucosamine pretreated group showed a significant reduction in the increased paw edema. Setnikar et al., 1991 showed that glucosamine protected against the edema provoked by carrageenan, In contrast, Singh et al., 2007 noticed that glucosamine caused a non-significant decrease of paw edema after carrageenan injection. Glucosamine produced a significant correction of carrageenan-induced hyper nociceptive response in parallel to Wen et al., 2010. Antinociceptive effect of glucosamine may be due to its anti-inflammatory effect; suppressing the raised IL-1, ROS, and neutrophil functions.
Atorvastatin pretreated group produced a significant reduction of carrageenan-induced paw edema following Jaiswal and Sontakke, 2012. atorvastatin treated group showed significant inhibition of the hyper nociceptive response following Santodomingo-Garzon et al., 2006 and Kamel et al., 2016
Atorvastatin inhibition of carrageenan-induced rat paw edema may be due to inhibition of prostaglandins and other inflammatory mediators such as IFN- γ, TNF-α, IL-1, and IL-6. Atorvastatin inhibits leukocyte-endothelial adhesion, reduces the levels of inducible nitric oxide synthase and inhibits the production of monocyte chemotactic protein-1. These anti-inflammatory effects are due to the inhibition of NF-kB that regulates the transcription of many inflammatory mediators and mutagenic signaling pathways (Baker et al., 2011). Statins also activate anti-inflammatory transcription factors known as peroxisome proliferator-activated receptors (PPARs) that interfere with NF-kB transcriptional activity (Cernuda-Morollón et al., 2002). Moreover, atorvastatin pretreatment showed correction of carrageenan-induced hyper nociception by inhibiting the production of proinflammatory mediators and deceased PGE2 sensitization action on nociceptors (Santodomingo-Garzon et al., 2006).
The pleiotropic effects of atorvastatin; including anti-inflammatory, analgesic, anti-catabolic, and antioxidant effects are assumed to have a beneficial role in the improvement of OA. The measurement of IL-1b and mmp-13 were performed to assess the anti-inflammatory and anticatabolic effects of atorvastatin. Besides, GSH assessment is used to detect the antioxidant effect of atorvastatin. Inflammation, oxidative stress are claimed to have a role in the catabolic state that occurs in OA. These pleiotropic effects are assumed to have a beneficial role in the improvement of OA. Also, it is reported that the expression of genes regulating cholesterol efflux is impaired in human OA chondrocytes, resulting in a toxic accumulation of lipid droplets in the chondrocyte that has a critical role in the development of OA (Tsezou et al., 2010). Atorvastatin has an anti-atheromatous effect that reduces structural deterioration of OA joints by improving the blood flow as the reduced blood flow in the small vessels in the subchondral bone may deteriorate the cartilage homeostasis and cause OA changes (Hoeven et al., 2013). Besides, atorvastatin is assumed to have an anabolic effect that is protective against cartilage damage (Karasawa, 2010). Atorvastatin may also inhibit osteoclasts activation by preventing mevalonate production, which leads to the loss of prenylation of small Ras and Rho GTPases and, consequently, disruption of downstream intracellular signaling pathways in osteoclasts (Hughes et al., 2007).
Conclusion :
The present study could present atorvastatin as a new useful potential DMOAD worse clinical trial for the treatment of OA. Accordingly, atorvastatin may be a promising DMOAD for treatment of OA especially in elderly patients suffering from hyperlipidemia, atherosclerosis.
Limitation of the studyOther markers which are assumed to be involved in the pathogenesis of OA have to be assessed. An assessment of other statins as a drug group has similar effects on OA like atorvastatin or not. Investigate the significance of atorvastatin cholesterol-lowering and anti-atheromatous action in correction subchondral bone ischemia. Also, an electron microscope examination of the affected joints may be of value to get a more precise evaluation.
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