INTRODUCTION:

Based on many early studies, statins have been shown to be effective in the treatment of skin lesions. Statins lower cholesterol levels, improve endothelial function, stabilize atherosclerotic plaques, and block inflammatory responses¹. Studies have shown that statins have beneficial immunomodulatory effects by reducing oxidative stress in cells and improving microvascular function². Additional clinical research is needed to assess the therapeutic effects of systemic and topical statins on wound healing. According to studies, treatment with rosuvastatin (RSV) beneficially alters neurovascular function and basal blood flow to the skin³. Despite a significant improvement in the healing process and a significant decrease in the lipid profile, it is unclear whether this effect, which improves blood flow and reduces the prevalence of neurodegenerative diseases, is the cause of these changes⁴.

As stated by a study,osteoanabolic and angiogenic factors, such as vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2, are expressed more frequently with statin use. The most potent osteoinductive factor, BMP-2, acts on immature mesenchymal cells, including osteoblasts, to induce osteogenesis.

BMPs are active osteoinductive substances. As an angiogenic cytokine, VEGF stimulates the production of bone metabolic factors by endothelial cells, which leads to the proliferation and differentiation of osteoblasts that results in the repair and regrowth of bones^5,6.

For diseases that cause bone loss, such as osteoporosis, for fractures, and for major bone abnormalities after severe trauma such as fractures and tumor removal, osteosynthetic drugs are the best treatment⁷. This musculoskeletal problem is closely related to the disease and physical injury. As 3-hydroxy-3-methylglutaryl coenzyme inhibitors, statins have been studied as chemicals that build bone. In in vitro and in vivo investigations, statins have been shown to stimulate bone growth and fracture repair in animals and humans⁸. Direct activation of TGF-b1 and BMP-2 and indirect reduction of osteoclast activity contribute to this effect. Studies have shown that statins have significant effects on bone remodeling processes after subcutaneous, oral and topical administration.

A lipid-lowering drug called rosuvastatin (RSV) is used to prevent cardiovascular disease. Based on simvastatin and atorvastatin, RSV has a long terminal life and powerful effects. In addition, this drug stimulates bone growth, promotes angiogenesis, and has anti-inflammatory effects. Clinical evaluations have been performed to determine the local effects of RSV on skeletal deformities based on clinical parameters^9,10. Statins contain a significant amount of initial hepatic metabolism, which leads to their limited systemic bioavailability. The systemic effects of RSV on bone formation should be studied using higher doses of RSV and tested in animal models to prevent adverse effects in humans and to determine the optimal dose. Histological methods can be used to evaluate animals for the studies, however, it may be unethical to obtain the patients of human material for histological analysis in clinical investigations¹¹. To our knowledge, few studies have focused on the systemic effects of RSV on skeletal abnormalities.The goal of this study was to investigate the possibility of systemic rosuvastatin (RSV) treatment at different time periods to assess the effects on bone remodeling and repair.

MATERIALS AND METHODS:

The Animal Use Ethics Committee concurred with this investigation. In this investigation, 20 mature male rabbits aged 3-1 years were used. Clinically healthy rabbits were housed in individual cages with 12/12 hours of light and an average weight of 3.0 kg. During the experiment, the rabbits were fed under conventional nutritional conditions, had free access to water, and were acclimatized to their environment for seven days prior to the experiment.

Two groups of rabbit used control and rosuvastatin group

Bone injury done

Each time points (day 7, day 14, day 21) Groups sacrificed for histology

1. Histology of bone using slide eosin-hematoxylin staining

2. Microscopy and Imaging

3. Scoring for inflammation and bone formation

RESULT:

The control group and the intervention group receiving rosuvastatin were used as the basis for the results of the follow-up trial. In addition, the results have been written three times. The results were first studied after seven days of implementation of the appropriate interventions. After fourteen days of analysis, a second analysis was performed and finally, a third intervention was performed after the experiment. All these analyses were performed according to the histological report of the bones which have been observed after the entire experiment.

The controlled group's results showed that there were no indications of alterations in the histophysiology seven days following the intervention. In line with this, the results of the intervention group showed that there were good findings that mildly indicated osteoprogenitor cells and moderate angiogenesis, which are stem cells that are crucial for bone growth and repair. These osteoprogenitor cells' existence signals bone repair at the damaged location.

On day fourteen of the trial, the following assessments were performed. The control group showed signs of angiogenesis and osteoblast development. This indicated the development of osteoblasts and the growth of new blood vessels. On the other hand, the results of the control group showed minimal to moderate bone growth. Angiogenesis had advanced and there was already significant bone growth there (Figure 1, Table 1).

Figure 1: Rosuvastatin-induced bone osteogenesis and suppressed inflammation.

The results of the bone assessment on day 21 showed slight bone formation and slight angiogenesis in the control group. In addition, there was a large number of osteoblasts in many areas at the beginning of aspiration, good angiogenesis in many areas, and reduced granulation tissue, but a low number of inflammatory cells. However, in the resulvastatin group, there was clearly a lot of inflammatory cell infiltration, a low number of fibroblasts, excessive angiogenesis, and a highly organized bone matrix with a large number of osteoblasts.

The results of the following experiment in general indicated that the group which received resulvastatin induced rapid regeneration of the bone as compared to the controlled group which represents an evident difference in both the groups and proves that resulvastatin is responsible for early bone remodeling and regeneration.

Table 1: Microscopic finding at tissue level on different timepoints.

Time points	Control	+Rosuvastatin
Day 7	-Only colt formation -No blood vessel formation -Very few fibroblast -No bone tissue formation. -Excessive inflammatory cell infiltration.	Very small new capillary formation (start of angiogenesis)and alter in granulation tissue with many fibroblast fine bone trabeculae formation in many area of tissue defect.
Day 14	-Many bone patch formation -Few blood vessels capillary formation -The amount granulation tissue decrease and the amount of inflammatory cells infiltration reduced.	Well development bone formation (thick bone trabeculae) with sufficient angiogenesis with skater granulation tissue and very few inflammatory cells (large no. of the osteoblast to osteocyte)
Day 21	-Many area in the suction start develop bone mass with large no of the osteoblast, good angiogenesis in the many area with decrease of granulation tissue. but there is little amount of inflammatory cell.	-Many inflammatory cells infiltration -Excessive blood vessel formation with dense highly organize bone matrix with extensive bone cell -Very few number of the fibroblast.

DISCUSSION:

The purpose of this study is to investigate the potential of the systemic administration of rosuvastatin (RSV) in different time frames on the healing of bones in the rabbits through oral route. The results represented that there is a clear difference between the two groups indicates that rosuvastatin is responsible for early bone remodelling and regeneration. The group that received rosuvastatin also experienced more fast bone regeneration than the control group.

These findings of the following experimental study are supported the results of the previously done study¹² which stated that As an important prerequisite for osteoblast production, reproduction, and differentiation, rosuvastatin encourages increased expression of BMP-2 in osteoblasts. It also aids in the healing of fractures and prevents the reoccurrence of fracture^13,14.

Statins are well known as lipid-lowering agents, but have also shown a variety of effects in experimental studies unrelated to lipid metabolism, including anti-inflammatory, antioxidant, immunomodulatory, proliferative, antithrombotic, and endothelial protective effects^15,16. Another study which is similar to the objective of the following experiment indicated added that A synthetic statin called rosuvastatin (RSV) has beneficial pharmacological properties such as minimal metabolism, hepatic selectivity, and increased inhibition of HMG-CoA reductase. The use of lipophilic statins, but not RSV, which, like pravastatin, is particularly hydrophilic compared to other statins, has been found to induce osteoblast differentiation in vitro¹⁷. To facilitate efficient entry in osteoblasts, there may be an active transport mechanism via solute carrier (SLC) transporters from the SLC16, SLC21/SLCO and SLC22 gene families. These transporters are in particular SLC16a1, Slco1a1, Slco2b1 and SLC22a8¹⁸.

According to the study RSV attenuated inflammation by reducing the expression of inflammatory cytokines such as interleukin 6 and tumor necrosis factor¹⁹. further determined that RSV alone or in combination with the antibiotic cefixime had antibacterial effects against Gram-positive and negative bacteria by reducing the minimal inhibitory concentration in insolation cultures²⁰.

CONCLUSION:

The purpose of the subsequent study was to evaluate the effects of rosuvastatin on osteogenesis and angiogenesis at the site of bone injury, as well as fracture repair and healing. According to the results of this study, early osteogenesis was much more prevalent in the rosuvastatin-treated group than in the control group. In addition, the study showed that rosuvastatin is necessary for effective bone regeneration and repair as well as early fracture healing. To further understand the effectiveness of RSV in the tissue healing process, more research is needed on the molecular components of the healing process when RSV is employed.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the College of Dentistry and College of Veterinary Medicine/University of Mosul for their provided facilities to accomplish this work.

REFERENCES:

1. Althanoon Z, Faisal IM, Ahmad AA, Merkhan MM, Merkhan MM. Pharmacological aspects of statins are relevant to their structural and physicochemical properties. Systematic Reviews in Pharmacy. 2020 Jul 1;11(7):167-71.

2. Almukhtar HM, Faisal IM, Merkhan MM. Short-term treatment with Atorvastatin selectively decreases Lymphocyte count. Research Journal of Pharmacy and Technology. 2022 Feb 1;15(2):689-94.

3. Almukhtar HM, Faisal IM, Merkhan MM. Acute effect of atorvastatin in comparison with rosuvastatin on glucose homeostasis in hypercholesteremic patients. Pharmacology. 2021;25:25-34.

4. Almukhtar HM, Faisal IM, Merkhan MM. Effects of statins on platelet count in hyperlipidemic patients. International Journal of Pharmaceutical Research. 2020 Apr;12(2):2640-4.

5. Younis HY, Thanoon IA, Fadhil NN, Merkhan MM. Effect of Zinc as an Add-On to Metformin Therapy on Glycemic control, Serum Insulin, and C-peptide Levels and Insulin Resistance in Type 2 Diabetes Mellitus Patient. Research Journal of Pharmacy and Technology. 2022 Mar 24;15(3):1184-8.

6. Merkhan MM, Abdullah E, Althanoon Z. Effect of Esomeprazole on serum creatinine and urea in patients with Peptic Ulcer. Research Journal of Pharmacy and Technology. 2022;15(1):160-4.

7. Al-Deresawi TS, Habeeb AA. The side effects of the Atorvastatin on liver of Hyperlipidemia Patients. Research Journal of Pharmacy and Technology. 2019 Sep 30;12(9):4310-2.

8. Al-Najeem HT, Al-Dujaili AN. Assessment of bone morphogentic protein receptor 2 level in pulmonary arterial hypertension disease. Research Journal of Pharmacy and Technology. 2017 Aug 28;10(8):2614-8.

9. Dhahir BM, Hameed IH, Jaber AR. Prospective and Retrospective Study of Fractures According to Trauma Mechanism and Type of Bone Fracture. Research Journal of Pharmacy and Technology. 2017 Nov 30;10(10):1827-35.

10. Subashree R, Arjunkumar R. Vitamin D deficiency in periodontal health. Research Journal of Pharmacy and Technology. 2014 Feb 28;7(2):248-52.

11. Strukov VI, Kislov AI, Eremina NV, Deriabina GP, Sergeeva-Kondrachenko MY, Antropov AY, Kuzmina YV, Tayrova KR, Petrova EV, Elistratov DG, Strukova-Jones OV. The use of Bone Tissue Non-Steroid Anabolizators in Treatment of Osteoporosis. Research Journal of Pharmacy and Technology. 2019 May 1;12(5):2195-9.

12. Ashna A, Jeena S, Vidhya PV, Venkateswaramurthy N, Sambathkumar R. Statins: Pleiotropic Effect. Research Journal of Pharmacy and Technology. 2016 Jul 1;9(7):977.

13. Gupta S, Jain R, Kapur S. Statins: A Review of Wonder Drug from Fungus. Research Journal of Pharmacy and Technology. 2014 Oct 28;7(10):1201.

14. Siddiqui MA, Roshan S. Pharmacokinetic Drug Interactions between HMG-COA Reductase Inhibitors Fluvastatin with Concomitantly administered Ticagrelor. Research Journal of Pharmacy and Technology. 2018;11(1):179-82.

15. Maged A, Mahmoud AA, Salah S, Abd-Elmonsif NM, Ghorab MM. Spray-dried rosuvastatin nanoparticles for promoting hair growth. AAPS PharmSciTech. 2020 Aug;21(6):1-2.

16. Wang BX, Li KP, Yu T, Feng HY. Rosuvastatin promotes osteogenic differentiation of mesenchymal stem cells in the rat model of osteoporosis by the Wnt/β-catenin signal. Eur Rev Med Pharmacol Sci. 2019 Nov 1;23(22):10161-8.

17. Shaheen MY, Basudan AM, Niazy AA, van den Beucken JJ, Jansen JA, Alghamdi HS. Impact of single or combined drug therapy on bone regeneration in healthy and osteoporotic rats. Tissue Engineering Part A. 2021 May 1;27(9-10):572-81.

18. Abraham S, Premnath A, Arunima PR, Kassim RM. Critical appraisal of bidirectional relationship between periodontitis and hyperlipidemia. Journal of International Society of Preventive & Community Dentistry. 2019 Mar;9(2):112.

19. Liu Y, Liu P, Song Y, Li S, Shi Y, Quan K, Yu G, Li P, An Q, Zhu W. A heparin–rosuvastatin-loaded P (LLA-CL) nanofiber-covered stent inhibits inflammatory smooth-muscle cell viability to reduce in-stent stenosis and thrombosis. Journal of nanobiotechnology. 2021 Dec;19(1):1-7.

20. Rajagopal A, ANAND M. The role of statins in periodontal therapy–a review. Cumhuriyet Dental Journal. 2022 Jun 30;25(2):200-5.

Received on 14.08.2022 Modified on 18.10.2022

Research J. Pharm. and Tech 2023; 16(6):2705-2708.

DOI: 10.52711/0974-360X.2023.00444