Induced arthritis, hepatocytes cytotoxicity and non-steroidal anti-inflammatory drugs (NSAIDs) – experimental study

This study aimed to histologically evaluate the pharmacological activity of meloxicam, COX-2 inhibitor, on liver function in arthritic rats. Meloxicam is a non-steroidal anti-inflammatory drug (NSAID), COX-2 inhibitor, generally prescribed in the area of rheumatology. Thus, 18 Wistar rats were divided into 3 groups with 6 animals each: G1 (Negative Control), G2 (Arthritis induced with Zymosan [Zy]), G3 (Arthritis induced with Zymosan [Zy] and treated with meloxicam). The G3 treatment started on the 21st day after induction. The animals were sacrificed, by overdose of Ketamine/Xylazine, seven, 14 and 21 days after the beginning of the meloxicam treatment. Liver samples were collected from the animals, fixed in Millonig buffer containing 10% formaldehyde and processed for conventional histological analysis. G1 animals showed absence of inflammation and degeneration of hepatocytes. In the G2 group, a slight cytoplasmic alteration was observed at seven and 14 days. At 21 days, intense cytoplasmic and nuclear disorganization was detected, accompanied by cell necrosis. In G3, large spacing between sinusoid capillaries was detected. In some 14-day-old animals, amyloidosis was observed, with the presence of a large number of vacuolated hepatocytes involved in an amorphous mass of necrotic cells. Most of the hepatocyte nuclei had compacted chromatin, evidencing cellular degeneration. Meloxican, used as an anti-inflammatory in the synovial region, showed that, in addition to its beneficial effects for the treatment of rheumatological diseases, it is capable of, when administered orally, promoting a high level of liver damage.


Introduction
Meloxicam, an enolcarboxamide whose chemical structure is 4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2benzothiazine-3-carboxamide-1,1-dioxide, is a new anti-inflammatory non-steroidal drug (NSAID), its pharmacokinetics vary according to the animal species, and may have a half-life of 3 to 36 hours. It is a preferred NSAID COX 2 inhibitor due to changes in its chemical structure in relation to other NSAIDs in its group. COX inhibitors not only interfere with inflammatory mechanisms, but also disrupt various body functions. Toxicity, gastrointestinal, renal failure, and cardiovascular risks are wellknown adverse effects of COX1 and COX 2 inhibitors (Nasr et al., 2020). These are the drugs (NSAIDs) most used to control pain and acute and chronic inflammation. They are largely organic acids indicated for the control of pain and acute and chronic inflammation. It is an NSAID preferentially COX 2 inhibitor due to changes in its chemical structure in relation to other NSAIDs in its group. These medications, non-steroidal anti-inflammatory drugs, make up a universe of 30% of the most used drugs with medical prescription or self-medication. COX inhibitors not only interfere with inflammatory mechanisms, but also disrupt various body functions. Gastrointestinal toxicity, kidney failure, and cardiovascular risks are well-known adverse effects of COX inhibitors (Sylvester, 2019).
Although COX has been identified for more than 20 years, it has been in recent years that it has become clear that there are two homologous isoforms, being referred to as COX-1 and COX-2 (Ju et al., 2022 ). In almost all normal tissues, the structural presence of COX-1 is detected, and in low to undetectable levels of COX-2, which can be expressed in greater amounts through the presence of cytokines, growth factors and tumor stimulants, suggesting its relevance. cancer and inflammatory processes. In this way, COX-1 was given the name constitutive; to COX-2, inductive (Samra et al., 2020). Recently, the existence of a third isoform of this enzyme family, called COX-3, was proposed, which, unlike COX-1 and COX-2, would not produce proinflammatory prostanoids, but anti-inflammatory substances. Such evidence can be seen in cases of chronic inflammatory diseases such as rheumatoid arthritis (Vane, 1996;Willoughby et al., 2000;Kummer & Coelho, 2002) Zymosan is derived from the cell wall of the fungus Saccharomyces cerevisiae, its main structural component is βglucan, which has immunostimulatory properties (Derbocio, 2005). Injection into the joint cavity causes an increase in vascular permeability leading to a cellular influx with a predominance of polymorphonuclear cells (Morsoleto, 2007). Zymosan is recognized by the dectin 1 receptor that is expressed in monocytes, macrophages, neutrophils and dendritic cells, after recognition it stimulates the production of inflammatory cytokines . This polysaccharide also causes the generation of anaphylatoxin C5a, through the activation of the alternative pathway of the complement system, with subsequent activation of monocytes and accumulation of neutrophils (Derbocio, 2005). The statistical treatment used was A Nova One Way with Turkey retest for significance level p ≤ 0.05. The program used to perform the test was Origin 7.0. The histological study was performed on slides stained with hematoxylin and eosin (H.E) with the aid of a light microscope**. Different parameters were defined for each organ, due to the peculiarity of each one. In the liver, the occurrence of:

Methodology
1 -Microvesicular steatosis -defined as the presence of lipid accumulation in the form of cytoplasmic microvesicles with a volume smaller than the nucleus; 2necrosischaracterized by condensation or effacement of the nucleus, intense cytoplasmic eosinophilia and Research, Society and Development, v. 11, n. 10, e526111033047, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i10.33047 destruction, loss of hepatocyte cord architecture (hepatocellular destrabeculation); 3apoptosischaracterized by the nuclear aspect, that is, the disposition of chromatin in the nucleus and by the appearance of the nuclear cytoplasm; 4leukocyte infiltrationpresence of leukocytes, especially neutrophils in the various parts of the hepatic lobule. In the liver, 50 fields were randomly observed from each animal under various magnifications (4 to 400x objectives). Lesions were graded in crosses ranging from absent (-) to ++++ depending on their intensity. These values were transformed into numbers for the application of statistical tests, as follows: Absence = 0; +/-= 0.5; + = 1; ++ = 2; +++ = 3 ++++ = 4.

Results
The animals of G1 (Figure 1), showed absence of inflammation and degeneration of hepatocytes as seen in Figures A   and B; in G2, a slight cytoplasmic and nuclear alteration was observed at seven and 14 days, as seen in the arrows in Figures C and E, respectively. At 21 days, there is intense cytoplasmic and nuclear degeneration accompanied by necrosis as seen in Figure   G. In G3, a large spacing between sinusoid capillaries can be observed ( Figure D). In this group, some 14-day-old animals presented amyloidosis as shown in figure F. In these cases, the hepatocytes are vacuolated and organized in an amorphous mass, with a large number of necrotic cells. Most of the hepatocyte nuclei had compacted chromatin, evidencing cellular degeneration.
At 21 days, the liver tissue is completely altered, showing disruption of its tissue morphology ( Figure H). At 21 days, the liver tissue is completely altered, showing disruption of its tissue morphology ( Figure H), Cheville, (1994). The analyzed results related to intralobular inflammatory infiltrates with seven days of treatment, showed P values ≤ 0.003 when relating G1 and G3 and P ≤ 0.001 for the G2 and G3 relationship. The groups with fourteen and twenty-one days did not present relevant statistical values.
After seven days of treatment, when analyzing the presence of necrosis, we can observe that the relationship between G1 and G2; G1 and G3 express a statistical value of P ≤ 0.05. With twenty-one days of treatment, we will find P statistical values ≤ 0.05 for the relationship between G1 and G3. The fourteen-day group did not present relevant statistical results. The fourteenday group did not present relevant statistical results. As Guicciardi and Gores (2005), report in their research.
The analyzed results related to intralobular inflammatory infiltrates with seven days of treatment, showed P values ≤ 0.003 when relating G1 and G3 and P ≤ 0.001 for the G2 and G3 relationship. The groups with fourteen and twenty-one days did not present relevant statistical values. Source: Authors.
Often the presence of infiltrating neutrophils close to apoptotic hepatocytes suggests an inflammatory response arising from an inflammatory response triggered by apoptosis Table 1. Regardless of the etiology, liver injury is usually characterized in the first phase by increased apoptosis of hepatocytes. This massive presence overwhelms the ability of phagocytic cells to remove dead cells. Autolysis of apoptotic bodies releases pro-inflammatory content.

Discussion
According to Pedroso andBatista 2017, Bhat et al., 2018;Bindu et al., 2020, continuous use of anti-rheumatic drugs causes potentially toxic effects, so a risk-benefit assessment should be made. The Ministry of Health, advised by the National Commission for the Incorporation of Technologies -CONITEC, recommends monitoring the use of these drugs through tests that assess, among other functions, the liver and kidney function. Based on several researches, our work aimed to evaluate liver function in arthritic rats after treatment with the NSAID meloxicam (Furst, 1997). Vane (1996) was the first to propose that the side effects of non-steroidal anti-inflammatory drugs resulted from the inhibition of the cyclooxygenase enzyme by these compounds. In a review of clinical cases by Maddrey et al (2000) involving 7400 patients, liver dysfunction occurred in 0.8% of those treated with celecoxib compared with 0.9% of those treated with placebo and 3.7% of those using diclofenac. of sodium.
According to O'Beirne and Cairns (2000) and Meunier, (2018), non-steroidal anti-inflammatory drugs, particularly sodium diclofenac, have been associated with severe hepatotoxicity. Recent case reports have shown that Celecoxib has been associated with the development of hepatitis and pancreatitis.
In a study carried out by Porta (2000), on autoimmune hepatitis, he shows important histological findings such as; chronic inflammatory infiltrates predominating in the portal, periportal and intralobular spaces, composed of lymphocytes, plasma cells and polymorphonuclear cells, enlargement of the portal spaces by fibrosis, derangement of the lobular architecture with lesions of the hepatocytes. These events which are corroborated by Parolin, and Reason, (2001).
In our study, several histological findings are shown with great similarity to those found in the relevant literature (Lida, 2005;Sriuttha, et al., 2018). Groups G2 and G3 have intralobular inflammatory infiltrates. Also in G3, in all experimental periods, cases of lobular disarrangement, cytoplasmic degeneration and disintegration (ballooning) and chromatin and nuclear disorganization are observed. In the animals analyzed after 21 days, areas of significant necrosis are observed. This morphological aspect is corroborated by (Cheville, 1994;Manov et al., 2006;Bindu et al., 2020).
Despite studies presented in recent years on cytotoxicity in hepatocytes caused by anti-inflammatory drugs in an arthritis model, there are still many doubts to be clarified, both in terms of mechanisms of action and criteria for use.

Conclusion
The experimental models used for induction and treatment with adverse substances such as Zymosam  according to the model proposed in this work, showed profound changes in the architecture of the liver tissue of the induced groups as described. In our study, the dosage of anti-inflammatory drug (meloxicam) seems to suggest an increase in necrosis in the liver tissue.
We suggest further research with tests that enable the analysis of tissue architecture and liver function. The lack of research on the subject, through the wide range of possible hepatotoxic agents, contributes to the large number of preventable liver diseases. New research will contribute to the reduction of such a large number of harm. Also suggest that researchers test new anti-inflammatory drugs, and include organs other than the liver. Such as organs of the digestive system and kidneys.