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Protective effects of harmine on Monosodium Iodoacetate-induced Osteoarthritis in rats: In vitro and in vivo studies
⁎Corresponding author at: Department of acupuncture and massage, Hangzhou Cancer Hospital, Hangzhou 310000, China. wujuecan@outlook.com (Juecan Wu)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Background
Osteoarthritis (OA) is a painful and debilitating disease, which is characterized by joint pain, swelling, restricted movement, and joint stiffness. It affects more than millions of people worldwide.
Objective
In this current work, we aimed to assess the beneficial roles of harmine on the monosodium iodoacetate (MI)-induced OA in rats.
Methodology
The in vitro studies were carried out in the LPS-induced RAW 264.7 cells and administered with 5 and 10 µM of harmine. In in vivo studies, 2.5 mg of MI diluted in 10 ml of saline (9%) was injected into the knee joints of the experimental rats to induce OA. The 25 and 50 mg/kg of harmine was treated one week before the MI injection and continued for 25 days after induction. The paw volume and arthritis score was measured after the completion of treatments. The levels of inflammatory cytokines, PGE-2, and NO in both RAW 264.7 cells and OA-induced rats using the assay kits. The MDA and antioxidants (GSH, CAT, and SOD) levels were assessed using the assay kits. The knee joint tissues were analyzed by histopathological study.
Results
The treatment with 5 and 10 µM of harmine effectively decreased the PGE-2 and NO levels in the LPS-exposed RAW 264.7 cells. The status of IL-6 and TNF-α also diminished by the harmine in the LPS-induced RAW 264.7 cells. The paw volume and arthritis score was decreased by the harmine treatment. The levels of PGE-2, IL-6, IL-1β, and TNF-α levels were depleted and IL-10 level was augmented by the harmine treatment in the OA-induced rats. The harmine are also suppressed the MDA and elevated the GSH, SOD, and CAT in the OA-induced rats. Further, the therapeutic roles of harmine also evidenced by the findings of the histopathological analysis.
Conclusion
The current research suggests that harmine is effective in attenuating OA in rats and significantly decreasing the OA-related oxidative stress and inflammation responses. The harmine also reduced the inflammatory response in the LPS-induced RAW 264.7 cells, therefore, it may be a talented agent for treating OA.
Keywords
Cytokines
Monosodium iodoacetate
Osteoarthritis
Prostaglandin-E2
RAW 264.7 cells
- OA
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Osteoarthritis
- MI
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Monosodium iodoacetate
- LPS
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Lipopolysaccharide
- ROS
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Reactive oxygen species
- MDA
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Malondialdehyde
- NO
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Nitric oxide
- GSH
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Glutathione
- CAT
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Catalase
- SOD
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Superoxide dismutase
- IL-6
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Interleukin-6
- TNF-α
-
Tumor necrosis factor-α
Abbreviations
1 Introduction
Osteoarthritis (OA), is a painful and debilitating disease characterized by chronic joint degradation that places a tremendous health burden and affects millions of people worldwide.
The main symptoms of OA include joint pain, joint swelling, restricted range of motion, and joint stiffness (Wang et al., 2016). The prevalence and cases stated in epidemiological research vary greatly since the data may be based on diverse diagnostic criteria, different joint sites, and varied patient demographics. Thus, the claimed OA prevalence ranged from 12.3 to 21.6% worldwide. (Jordan et al., 2014). Women are more likely than males to acquire OA. Additionally, the prevalence of OA varies greatly between different geographical places (such as developing or developed nations, and rural or metropolitan areas) (Cross et al., 2014). OA affects the entire joint, affecting almost all of the joint tissues as a result of the degenerative process (Robinson et al., 2016). Anabolic and catabolic substances generated by chondrocytes are metabolically out of balance in OA, which causes cartilage to degrade and break down. The participation of the synovium, infrapatellar fat pad, and other joint tissues, which produce inflammatory mediators, is well recognized. The increase in joint loading is also influenced by modifications to the subchondral bone and muscles (Ansari et al., 2020).
Oxidative stress is a major contributor to the pathophysiology and development of OA. The dysregulation between excessive production of oxidative stress markers such as reactive oxygen species (ROS), malondialdehyde (MDA), and nitric oxide (NO) and their scavenging by antioxidants such as glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) leads to excessive oxidative stress. The oxidative stress damages nuclear and mitochondrial DNA at the cellular level, which affects protein transcription and cell signaling pathways (Marchev et al., 2017; Dranitsina et al., 2019; Dranitsina et al., 2018). Excessive oxidative stress triggers cells in the synovium, which contributes to the main signs of OA (Bolduc et al., 2019). Additionally, OA is linked to varying degrees of synovial inflammation that can be brought on by the breakdown and malfunction of the cartilage (Wang et al., 2021). Reactive oxygen species (ROS), cytokines, chemokines, and proteases are all produced by macrophages, synovial fibroblasts, and chondrocytes as OA progresses. The inflammatory cytokines increase synovitis, impair chondrocyte viability, and stimulate the synthesis of several proteases that can deteriorate the cartilage matrix (Grassel et al., 2021).
Along with the degeneration of cartilage structures, the etiology of OA also involves the remodeling of subchondral bones, hypertrophy of articular cartilage, and synovial inflammation (Mobasheri et al., 2017). The innate immune system over production of inflammatory cytokines such as interleukins (ILs) and tumor necrosis factor-α (TNF-α) has a major role in mediating the moderate and chronic inflammation associated with OA (Griffin and Scanzello, 2019). The main treatments for OA are non-steroidal anti-inflammatory medications (NSAIDs), in addition to physiotherapy and exercise. But persistent systemic drug administration has been linked to serious adverse effects, including osteoporosis, an elevated risk of cardiovascular disease, and gastrointestinal issues (Kloppenburg and Berenbaum, 2020). The primary goals of most therapeutic therapies are pain management, functional enhancement, and artificial joint replacement. In order to lubricate joints and encourage bone healing, several molecular treatments, such as the intra-articular injection of hyaluronic acid polymer, are frequently employed in the treatment of osteoarthritis. This slows the growth of arthritis and increases joint mobility, but it does not prevent the disease (Maudens et al., 2018). These therapies have known harmful side effects and toxicity, and they only offer transient systemic relief (King et al., 2020).
When non-surgical treatments are ineffective at managing OA symptoms, joint replacement is advised. But OA cannot be cured by joint replacement; one year after surgery, 20–30% of patients who have knee and hip replacements report little to no improvement and are dissatisfied with the results (Jones et al., 2007). This is true even though researchers are still trying to better understand the factors that contribute to poor outcomes after knee/hip joint replacement. Worldwide, there is still a pressing need to identify efficient and secure non-surgical techniques for the successful treatment of OA. The identification of novel natural substances is crucial for the progression of new drugs for OA treatment with high potential and lesser aftereffects.
Harmine, a naturally occurring β-carboline is extensively found in the Peganum harmala L. (Patel et al., 2012). Harmine has several biological properties, including those against bacteria, fungi, free radicals, and cancer (Prasad Kushwaha et al., 2019). Additionally, harmine is a perfect therapeutic candidate for the treatment of liver cancer since it selectively damages liver cancer cells while having few negative side effects on healthy liver cells (Zhang et al., 2015). By focusing on apoptosis, autophagy, aberrant cell growth, and metastasis, harmine has remarkable anticancer capabilities (Jalali et al., 2021). According to studies, harmine possesses outstanding anticancer properties that inhibit the growth, metastasis, and invasion of breast and liver cancer cells while inducing apoptosis and autophagy (Geng et al., 2018; Chen et al., 2022; He et al., 2022). A recent study found that harmine reduces cardiac hypertrophy (Huang et al., 2022), neurotoxicity (Habib et al., 2022), and sepsis-induced cardiac dysfunction (Ruan et al., 2021). A previous study also reported the in vitro chondrogenic effects of harmine (Guzman et al., 2003). In the current study, we planned to investigate the therapeutic potential of harmine against OA in rats.
2 Materials and methods
2.1 Chemicals
All the chemicals including harmine and MI were obtained from Sigma-Aldrich, USA. The assay kits were obtained from BioVision, Thermofisher scientific, and Elabscience (USA) for the measurement of biochemical parameters. All chemicals were utilized in an analytical grade.
2.2 In vitro studies
2.2.1 Collection and maintenance of cells
The macrophage-like RAW 264.7 cells were collected from the ATCC, USA, and grown on DMEM medium enriched with FBS (10%) and antimycotic mixtures (1%) at 37 °C for 24 hr in a moistened and CO2 (5%) provided incubator. After reaching 80% confluency, cells were trypsinized and used for assays.
2.2.2 In vitro anti-inflammatory activity of harmine on RAW 264.7 cells
The RAW 264.7 cells (ATCC No. TIB-71) were grown on the 96-wellplate with DMEM medium at 5 × 103 cell population per well for 24 h at 37 °C. Then, the cells were administered with 5 and 10 µM of harmine with or without 200 ng/ml of LPS for 24 h at 37 °C. After the completion of treatments, the cells were trypsinized and then cell pellets were homogenized using saline solution to prepare the cell lysates. The cell lysate was obtained by centrifuging the homogenized mixture at 2000 rpm for 10 min and supernatant was stored in 4 °C until further use.
2.2.3 Quantification of PGE-2, NO, and proinflammatory cytokines
The obtained cell lysates were used to quantify the levels of NO (#MBS1608286)), PGE-2 (#MBS262150), TNF-α (#MBS825075), and IL-6 (#MBS2021530) by using the corresponding assay kits. All the assays were conducted using marker-specific assay kits in accordance with recommended protocols of the manufacturer (MyBioSource, USA). All the assays were performed in triplicates.
2.3 In vivo studies
2.3.1 Experimental rats
A 6–8 weeks old healthy Wistar albino rats in good health weighing around 210 ± 20 g were obtained form institutional animal house. The rats were kept in clean polypropylene cabins with a temperature of 22–26 °C, air moisture of 50–60%, and a 12-hour light/dark series. The current study was approved by the institutional animal ethics committee. Before beginning the trials, the rats spent a week becoming acclimated in the lab.
2.3.2 Experimental groups and treatment methods
All of the rats were divided into four groups of six each. Group I was used as the control. In order to induce the OA, 2.5 mg of MI diluted in 10 ml of saline (9%) was injected into the right and left knee joints with help of a sterile needle (Group-II) (Hara et al., 2013). As indicated in group-II, the rats underwent OA induction after receiving 25 and 50 mg/kg of harmine for one week and the treatment was continued each day for 25 days (Group III and IV). The rats were sacrificed by cervical dislocation under anesthesia (90 mg/kg of ketamine) when the treatments were finished, then blood and joint tissue samples were taken for additional biochemical study.
2.3.3 Measurement of paw volume
Using a plethysmometer, the level of the hind paws of the experimental and control rats was measured from the start and end of the investigations. The changes in the paw volume of the control and treated rats were identified, and values are recorded.
2.3.4 Determination of arthritis score
To estimate the severity and grade of the disease, the arthritis score of the control and treated rats were calculated. According to the severity of the condition, the rats' arthritic scores were calculated using a 25-point scoring system.
2.3.5 Quantification of PGE-2 and NO levels
Using specific test kits, the contents of PGE-2 (ab287802) and NO (ab65328) in control and experimental rats were examined using the manufacturer's instructions (BioVision, USA). Each test was carried out in triplicates.
2.3.6 Measurement of inflammatory cytokines
The assay kits were utilized to measure the status of IL-6 (E-EL-R0015), IL-10 (E-EL-R0016), IL-1β (E-EL-R0012), and TNF-α (E-EL-RB0011) in the serum of control and experimental rats. The manufacturer's suggested protocols were used to conduct the assays (Elabscience, Houston, USA). There were three duplicates of each experiment.
2.3.7 Determination of oxidative and antioxidative biomarkers in the experimental rats
The joint tissues collected from the rats were centrifuged at 10,000 rpm for 15 min after being homogenized with saline solution. The levels of oxidative stress and antioxidant indicators such as MDA (E-EL-0060), CAT (E-BC-K031-M), SOD (E-EL-R1424), and GSH (E-EL-0026) were measured in the supernatant using the corresponding test kits. Following the manufacturer's suggested guidelines, each experiment was carried out three times (Elabscience, Houston, USA).
2.3.8 Histopathological analysis of knee joint tissues
The joint tissues were removed following the animal sacrifices for histopathological analysis. After processing the tissues with 10% formalin, the 20% formic acid solution was used to decalcify the joint tissues. The joint tissues were decalcified, entrenched in paraffin wax to construct tissue blocks, and then cut into 5 µm diameter. Hematoxylin and eosin were then utilized to stain the tissue slices. Finally, a light microscope was used to analyze the histological alterations in the hind limbs of experimental rats.
2.4 Statistical analysis
GraphPad Prism software was used to statistically evaluate the results that were produced. The final values were illustrated as the mean ± SD of measurements made in three duplicates. The variances between groups were measured using the one-way ANOVA and Duncan's multiple range test (DMRT), with p < 0.05 being considered significant.
3 Results
3.1 Effect of harmine on the PGE-2 and NO levels in the LPS-exposed RAW 264.7 cells
Fig. 1 shows the levels of PGE-2 and NO in the cell lysates of control and exposed RAW 264.7 cells. The LPS-exposed RAW 264.7 cells revealed a considerable elevation in the levels of PGE-2 and NO. However, the 5 and 10 µM of harmine treatment considerably depleted the PGE-2 and NO levels. These results showed that harmine treatment to the LPS-exposed RAW 264.7 cells considerably diminished inflammatory markers (Fig. 1).
Effect of harmine on the PGE-2 and NO levels in the LPS-induced RAW 264.7 cells: Each bar displays the mean ± SD of the values obtained in triplicate from each assay. Using the Graphpad Prism software, all of the data were statistically analyzed using one-way ANOVA and Dunnett's post hoc analysis. Note that “#” denotes statistical significance at p < 0.01 for the control group and “*” denotes statistical significance at p < 0.05 for the OA-induced group. Group I: Control; Group II: MI-induced OA rats; Group III: OA rats treated with 25 mg/kg of harmine; Group IV: OA rats treated with 50 mg/kg of harmine.
3.2 Effect of harmine on the IL-6 and TNF-α levels in the LPS-exposed RAW 264.7 cells
The IL-6 and TNF-α status was investigated in both the control and harmine-administered RAW 264.7 cells, and the results were shown in Fig. 2. The increased IL-6 and TNF-α levels were observed on the LPS-induced RAW 264.7 cells than in the control. However, the treatment with 5 and 10 µM of harmine treatment substantially decreased the IL-6 and TNF-α levels in the LPS-exposed RAW 264.7 cells (Fig. 2). These outcomes reveal that in the RAW 264.7 cells, the harmine inhibits the pro-inflammatory cytokine levels, which indicate it’s in vitro anti-inflammatory property.
Effect of harmine on the IL-6 and TNF-α levels in the LPS-induced RAW 264.7 cells: Each bar displays the mean ± SD of the values obtained in triplicate from each assay. Using the Graphpad Prism software, all of the data were statistically analyzed using one-way ANOVA and Dunnett's post hoc analysis. Note that “#” denotes statistical significance at p < 0.01 for the control group and “*” denotes statistical significance at p < 0.05 for the OA-induced group. Group I: Control; Group II: MI-induced OA rats; Group III: OA rats treated with 25 mg/kg of harmine; Group IV: OA rats treated with 50 mg/kg of harmine.
3.3 Effect of harmine on the arthritis score and paw volume in the experimental rats
When compared to the control group, the OA-induced rats had a striking rise in the paw volume and arthritis score, as shown in Fig. 3. However, the harmine at dosages of 25 and 50 mg/kg substantially reversed these modifications, as seen by the lowered arthritic score and paw volume in the OA-induced rats (Fig. 3). The harmine at a higher dose significantly reduced the arthritic symptoms in the rats as compared to 25 mg/kg, which was supported by the harmine's effective reducing activity at 50 mg/kg.
Effect of harmine on the arthritis index score and hind paw volume in the experimental rats: Each bar displays the mean ± SD of the values obtained in triplicate from each assay. Using the Graphpad Prism software, all of the data were statistically analyzed using one-way ANOVA and Dunnett's post hoc analysis. Note that “#” denotes statistical significance at p < 0.01 for the control group and “*” denotes statistical significance at p < 0.05 for the OA-induced group. Group I: Control; Group II: MI-induced OA rats; Group III: OA rats treated with 25 mg/kg of harmine; Group IV: OA rats treated with 50 mg/kg of harmine.
3.4 Effect of harmine on the PGE-2 level in the experimental rats
In OA-induced rats, harmine's lowering effects on PGE-2 level were investigated; the outcomes are revealed in Fig. 4. The OA-induced rats revealed a substantial increase in PGE-2 level. Meanwhile, the PGE-2 level of OA-induced rats was significantly lowered by the 25 and 50 mg/kg of harmine treatments (Fig. 4). These results validated harmine's therapeutic benefits for OA-induced rats.
Effect of harmine on the PGE-2 level in the experimental rats: Each bar displays the mean ± SD of the values obtained in triplicate from each assay. Using the Graphpad Prism software, all of the data were statistically analyzed using one-way ANOVA and Dunnett's post hoc analysis. Note that “#” denotes statistical significance at p < 0.01 for the control group and “*” denotes statistical significance at p < 0.05 for the OA-induced group. Group I: Control; Group II: MI-induced OA rats; Group III: OA rats treated with 25 mg/kg of harmine; Group IV: OA rats treated with 50 mg/kg of harmine.
3.5 Effect of harmine on the pro-inflammatory cytokines level in the experimental rats
Fig. 5 illustrates the findings of the effects of harmine on the status of inflammatory mediators. The OA-induced rats showed considerably lower levels of IL-10 and significantly higher levels of IL-6, IL-1β, and TNF-α as compared to the control group. Significantly, the OA-induced rats' IL-6, IL-1β, and TNF-α status was dramatically reduced by the 25 and 50 mg/kg harmine treatments, whereas their IL-10 levels were elevated (Fig. 5). These results demonstrated the harmine's ability to reduce inflammation.
Effect of harmine on the pro-inflammatory cytokines level in the experimental rats: Each bar displays the mean ± SD of the values obtained in triplicate from each assay. Using the Graphpad Prism software, all of the data were statistically analyzed using one-way ANOVA and Dunnett's post hoc analysis. Note that “#” denotes statistical significance at p < 0.01 for the control group and “*” denotes statistical significance at p < 0.05 for the OA-induced group. Group I: Control; Group II: MI-induced OA rats; Group III: OA rats treated with 25 mg/kg of harmine; Group IV: OA rats treated with 50 mg/kg of harmine.
3.6 Effect of harmine on the oxidative and antioxidative biomarkers in the joint tissues of experimental rats
The antioxidant properties of harmine were evaluated by examining the effect of harmine on the variations in MDA, SOD, CAT, and GSH in the joint tissue homogenates. As shown in Fig. 6, the joint tissue homogenate of OA-induced rats had significantly higher MDA levels and lower status of CAT, SOD, and GSH than the control group. It's interesting to note that in the joint tissue homogenates of OA-induced rats, harmine at 25 and 50 mg/kg substantially suppressed the MDA level and augmented GSH, SOD, and CAT (Fig. 6). These findings demonstrated the anti-oxidant property of harmine.
Effect of harmine on the oxidative and antioxidative biomarkers in the joint tissues of experimental rats: Each bar displays the mean ± SD of the values obtained in triplicate from each assay. Using the Graphpad Prism software, all of the data were statistically analyzed using one-way ANOVA and Dunnett's post hoc analysis. Note that “#” denotes statistical significance at p < 0.01 for the control group and “*” denotes statistical significance at p < 0.05 for the OA-induced group. Group I: Control; Group II: MI-induced OA rats; Group III: OA rats treated with 25 mg/kg of harmine; Group IV: OA rats treated with 50 mg/kg of harmine.
3.7 Effect of harmine on the knee joint tissue histopathology of experimental rats
The results of the histopathological analysis of the knee joint tissues in the experimental rats are shown in Fig. 7. The control rats showed normal structures. The OA-induced rats showed considerable matrix bone loss, chondrocyte hyperplasia, and cartilage deterioration. However, in OA-induced rats, harmine at dosages of 25 and 50 mg/kg dramatically suppressed tissue injury, cartilage degradation, and loss of cartilage matrix (Fig. 7).
Effect of harmine on the knee joint tissue histopathology of experimental rats: The control rats showed normal structures (Group I). The knee joint tissues of OA-induced rats revealed considerable matrix bone loss (black arrow), collapsed cartilage surface (blue arrows), and hyperplasia of chondrocyte cells (yellow arrows) (Group II). The harmine at doses of 25 and 50 mg/kg dramatically reduced tissue injury, cartilage degradation, and loss of cartilage matrix in the joint tissues of OA-induced rats (Group III & IV, respectively). Scale bar: 50 µm; magnification: 40 ×.
4 Discussion
OA is a common degenerative bone disease, which affects millions of people worldwide, and is characterized by the gradual destruction and loss of articular cartilage and periarticular muscles (Sirse, 2022). In order to restore joint functionality in patients with advanced OA, joint replacement surgery is required. A patients' quality of life is negatively impacted by this, which imposes a significant social and financial burden. The knee joints are the most frequently afflicted by OA, followed by the hands and hips (Favero et al., 2022). The search for a new, effective treatment for the OA condition is the researcher's primary research goal. Researchers focus on natural remedies and plant-based medications for the treatment of OA, as they have greater beneficial effects and fewer adverse effects (Jalalpure et al., 2011). Here, we assessed the beneficial roles of harmine in OA-induced rats. The epidemiology and ethnic diversity of this disease are dynamic worldwide. Compared to men, women are more susceptible to this disease (Di et al., 2011). It is still difficult to pinpoint the exact cause of OA and the risk factors that contribute to its rapid progression.
Edema is caused by tissue damage, which is followed by the migration of mast cells, macrophages, and leukocytes as well as the extravasation of tiny blood vessels (Allam and Anders, 2008). In the OA paradigm, the edematous paw swelling is caused by increased amounts of prostaglandins, particularly PGE-2. Paw edema is also related to increased fluid output in the inflamed area, the penetrability of the vascular system, and cellular penetration. A popular technique for determining the potential of anti-arthritic candidates is to measure the diameter of the paw. A major indicator of a drug's ability to combat inflammation is the reduction in paw diameter, which shows lower production of inflammatory mediators (Bose et al., 2014). An indicator of the severity of OA, the arthritic score measures joint inflammation (Choudhary et al., 2014). Harmine has anti-inflammatory capabilities, as evidenced by the fact that harmine-administered rats had much lesser arthritic scores than OA-induced rats. Paw swelling can be sensitively and quickly measured to determine the level of inflammation and therapeutic potential of medications. OA-induced rats revealed increased paw volume, which was consistent with previously published findings (Kshirsagar et al., 2014). However, giving harmine to OA-treated rats reduced the paw swelling. This finding demonstrates the anti-inflammatory properties of harmine against OA-triggered paw inflammation.
Oxidative stress and the development of OA are also closely related. Higher ROS accumulation in OA chondrocytes and cartilage may be triggered by oxidative stress (Zahan et al., 2020; Korotkyi et al., 2020). The pathophysiology of OA is heavily influenced by oxidative stress, which aggravates inflammation. Cellular proteins, DNA, and lipids are affected by the interactions of free radicals produced during inflammation (Chu et al., 2021; Korotkyi et al., 2019). MDA is produced as a result of free radicals, and then triggers the generation of mediators that cause inflammation. The body has an inbuilt antioxidant system that causes the free radicals to be neutralized, including GSH, CAT, and SOD (Shal et al., 2020). The harmine considerably reduced oxidative stress indicators and increased antioxidants. The new study's findings are in line with those of an earlier study (Quinonez-Flores et al., 2016). Comparing OA-induced rats to the healthy control, a decrease in SOD, GSH, and CAT status was seen. This could be a result of the overproduction of free radicals, which inhibit the action of antioxidants (Yousef et al., 2009). In the current study, higher MDA was observed in the joints of OA-induced rats, compared to normal rats, indicating an elevation in oxidative stress. This could be a result of the intracellular antioxidant defense mechanism being weakened in OA, which leaves more free radicals to scavenge, leading to higher oxidative stress (Mititelu et al., 2020).
It was reported that the pathophysiology and development of OA are significantly influenced by inflammatory cytokines and local inflammatory responses (Tu et al., 2020). Inflammation is a crucial player in OA progression and facilitates bone destruction (Tatiya et al., 2017). TNF-α, which promotes synovial fibroblasts, leukocyte migration, and cellular adhesion molecules into the injured joint, intensifies the inflammatory response (Ouyang et al., 2019). TNF-α stimulates proinflammatory cytokines and inhibits anti-inflammatory mediators like IL-10 to further worsen the inflammation. IL-1β facilitates the breakdown of cartilage, and bone resorption, and changed the synthesis of PGE2 and NO (Singh et al., 2021). IL-6 stimulates inflammation by causing blood vessels to expand further. When exposed to IL-6, inflammatory cells are encouraged to proliferate and differentiate, which also plays a critical function in inflammatory disorders. IL-6, IL-1β, and TNF-α are therefore utilized as a measure of the effectiveness of anti-inflammatory drugs (Takeuchi et al., 2021). In the early stages of OA, elevated amounts of these cytokines can be found in the chondrocytes and synoviocytes, which is a key mediator of joint inflammation (Molnar et al., 2021). In addition, the levels of IL-6 and TNF-α are aberrantly augmented in the cartilage and synovial fluids of OA patients, suggesting that they are vital players in OA progression (de Lange-Brokaar et al., 2012). In order to stop or halt the OA development, it may be useful to suppress the inflammatory response. Here, we observed that the OA-induced rats revealed higher levels of IL-6, IL-1β, and TNF-α while the reduced level of IL-10 level. Interestingly, harmine treatment substantially depleted the pro-inflammatory cytokines and augmented the IL-10 in the OA-induced rats. Furthermore, harmine treatment also depleted the IL-6 and TNF-α status in the LPS-exposed RAW 264.7 cells. These outcomes witnessed the anti-inflammatory properties of the harmine on OA-induced rats.
PGE2 serves as a crucial second messenger in the inflammation process. The degree of PGE2 expression is directly correlated with the inflammatory response. High levels of PGE2 have been proven to expand capillaries, increase vascular permeability, cause edema and tissue congestion, degrade cartilage, and bone breakdown (Takeuchi et al., 2020). It is well known that NO mediates synovial inflammation. Excessive NO production can trigger chronic inflammatory disorders, including OA. Experimentally induced arthritic animals revealed a significant rise in NO levels (Rovensky et al., 2009). The PGE2 and NO may accelerate the progression of OA by increasing the expression of proteases and decreasing the synthesis of macromolecules (Wu et al., 2020). Therefore, examining the levels of PGE2 and NO can aid in determining the inhibition of inflammatory conditions in rats. We found that the OA-induced rats revealed a considerable increment in the NO and PGE-2 levels. Surprisingly, our study's findings revealed that harmine might limit the overproduction of NO and PGE2 in the OA-induced rats as well as LPS-exposed RAW 264.7 cells. These outcomes revealed the anti-inflammatory properties of the harmine on OA-induced rats.
Histological examinations are essential for determining the pathological alterations that occur in an inflammatory environment, such as inflammatory cell infiltration, edema, and fibrotic changes (Feldmann and Maini, 2008). In the current investigation, harmine considerably improved the histological alterations in the knee joints of OA-induced rats. Improvement in joint tissue histological characteristics has been linked to a decrease in OA condition, which is in line with an earlier study by Ansari et al. (Ansari et al., 2021). These findings revealed the therapeutic actions of harmine on OA-induced rats.
5 Conclusion
The current research suggests that harmine is effective in attenuating OA in rats by significantly reducing the OA-related oxidative stress and inflammation responses. Harmine treatment effectively decreased the PGE-2, NO, and inflammatory marker levels in both OA-induced rats as well as LPS-exposed RAW 264.7 cells. The harmine also decreased MDA and elevated the CAT, SOD, and GSH levels. The harmine treatment also averted bone loss and damage, as shown by the histological results. As a result, it may be a candidate for treating OA. To confirm the precise therapeutic mechanism of harmine, additional research is still required in the future (Fig. 8).
Proposed mechanisms of harmine on LPS-induced RAW 264.7 cells and osteoarthritis-induced rats.
Acknowledgment
This project was supported by Researchers Supporting Project number (RSPD2023R712), King Saud University, Riyadh, Saudi Arabia.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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