Orientador: Prof. Dr. Jurandir Fernando Comar

 Data da Defesa: 13/08/2019

Quercetin is one of the most abundant flavonoids in the diet and has been reported to present many antioxidant and anti-inflammatory properties. It is a potent depurator of free radicals and it is therefore largey utilized as antioxidant in nutritional supplements with the purpose of preventing aging-associated diseases. It presents antioxidant activity in vitro higher than endogenous antioxidants and the supplementation with this flavonoid has been shown to increase the plasmatic antioxidant capacity, as well as improvement in inflammation of rats with adjuvant-induced arthritis. High concentrations of quercetin improve inflammation in rats with arthritis, but have been associated with pro-oxidant and mitochondriotoxic actions. Quercetin carried in drug delivery systems orally administered at low doses improves hepatotoxicity and oxidative stress in rats livers, an action that does not occur with free quercetin. In this regard, many strategies have been used to improve the oral bioavailability of quercetin, such as quercetin-loaded biodegradable polymers-based micro and nanoparticles.
To evaluate whether long-term treatment with microencapsulated quercetin at a low dose of 10 mg • Kg-1 attenuates the oxidative stress in the liver and brain of rats with adjuvant-induced arthritis.
For arthritis induction, Male Holtzman rats (180g) were subcutaneously injected in the left hind paw with 0.1 mL (500 μg) of Freund's adjuvant (heat inactivated Mycobacterium tuberculosis).
Thirty animals were randomly distributed into five groups: Controls (C); Controls (C + Q) treated with microencapsulated quercetin at a dose of 10 mg • Kg-1; Arthritic rats (A); Arthritic rats (A + Q) treated with microencapsulated quercetin at a dose of 10 mg • Kg-1; and arthritic rats (AIBU) treated with ibuprofen at a dose of 20 mg • Kg-1. The animals were treated for 60 days.
Rats fasted (12 h) were anesthetized with an overdose of sodium thiopental (100 mg·kg-1) and the brain immediately removed, freeze-clamped in liquid nitrogen. The peritoneal cavity was exposed, the liver was removed and divided into two parts: one was immediately freeze-clamped and stored in liquid nitrogen for oxidative status assessment and the other was used for mitochondria isolation. Total homogenate was used to determine the levels of lipoperoxides (TBARS) and reduced glutathione (GSH). An aliquot of total homogenate was separated, centrifuged and the supernatant obtained after centrifugation was used to determine: protein carbonyl groups, ROS content, the activities of catalase and superoxide dismutase. All these determinations were estimated by spectrophotometry or spectrofluorimetry.
Hepatic mitochondria were isolated by differential centrifugation. Mitochondrial oxygen consumption was measured polarographically using a Teflon-shielded platinum electrode. Succinate and α-ketoglutarate were used as electron donor
substrates for complexes I and II, and ADP was added at appropriate times. Mitochondrial fraction was used to determine oxygen consumption and mitochondrial membrane energization. The activities of NADH-oxidase and succinate-oxidase were measured polarographically using freeze-thawing disrupted mitochondria. The rate of mitochondrial ROS production (real-time ROS production) was estimated by measuring the linear fluorescence increase.
The results show a decrease in oxidative stress from the reduced levels of TBARS, protein carbonyl groups and reactive oxygen species (ROS) in the liver and brain of arthritic rats orally treated with quercetin-loaded microcapsules.
The treatment of control rats with microencapsulated quercetin did not modify the levels of GSH, but completely reestablished their levels in the liver and brain of arthritic rats. The levels of TBARS, protein carbonyl and GSH were not improved in the arthritic rats treated with ibuprofen.
Treatment of arthritic rats with microencapsulated quercetin did not increase the activity of catalase of both liver and brain. Treatment of arthritic rats with ibuprofen improved the catalase activity in the liver, but did not modify the enzyme activity in the brain. The SOD activity was lower in the liver of arthritic rats trated with empty microcapsules, and the treatment with both quercetin and ibuprofen did not improve the enzyme activity.
No modification in the respiratory parameters were verified in the hepatic mitochondria of all groups, except by a slightly increase in the ADP/O ratio of –non-treated and treated arthritic rats when succinate was the respiratory substrate. Similarly, no modifications were observed in the mitochondrial ROS generation. Regarding ruptured mitochondria, the treatment of arthritic rats with microencapsulated quercetin completely restored the activity of succinate oxidase, but did not modify the TMPD/ascorbate-stimulated oxygen consumption.
In summary, our results show that quercetin was efficiently encapsulated inside the pectin/casein polymeric matrix and long-term treatment with quercetin microcapsules orally administrated at the dose of 10 mg·Kg-1 improved the oxidative stress in the brain and liver of rats with adjuvant-induced arthritis. In addition, quercetin was neither hepatotoxic nor mitochondriotoxic, and the anti-inflammatory activity already reported to the compound was not verified in arthritic rats.
It can be concluded that the pectin/casein microcapsules loaded with quercetin may modify oxidative stress independently of the anti-inflammatory activity and can be used as an antioxidant supplement in the treatment of rheumatoid arthritis.
Keywords: adjuvant-induced arthritis, liver oxidative status, quercetin-loaded microparticles, pectin/casein microparticles.


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