Qom University of Medical Sciences Journal

Introduction
Nonalcoholic Steatohepatitis (NASH) is one of the common liver diseases, characterized by excessive lipid accumulation (steatosis) and inflammation in the liver. Epidemiological studies have shown that almost 20% of patients with nonalcoholic fatty liver disease have NASH. It is predicted that NASH will become the main reason for liver transplantation from 2020 to 2025. NASH can lead to cirrhosis, fibrosis and ultimately hepatocellular carcinoma. Although the pathophysiology of NASH is not completely understood, studies have demonstrated that inflammation is one of main reasons for this disease which takes place from different pathways including matrix metalloproteinases and prostaglandins. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandins. It stimulates prostaglandin E2 (PGE2), which, with increasing inflammation, can cause the development of NASH. Evidence shows that COX2 and prostaglandin E2 levels are high in people with NASH. Increased prostaglandin E2 level can increase the production of Matrix Metallopeptidase 9 (MMP-9) through the effect on EP2 and EP4 prostaglandin receptors in cells.
Rosmarinic Acid (RA) is a pure compound which is commonly found in plants from Lamiaceae family including Ros-marinus officinalis (rosemary), Coleus aromaticus Origanum vulgare L. (oregano) and thymus vulgaris L. (thyme). The beneficial effects of RA on the diabetes, sepsis, hepatotoxicity and brain injuries have already been reported. It reduces blood glucose by inhibiting insulin resistance and increasing GLUT4 expression. It is considered as an antioxidant and anti-inflammatory product with positive effects on liver injuries. This study aims to evaluate the effect of RA on the MMP-9 expression level and COX-2/PGE2 pathway in male mice with NASH.
Methods
In this study, samples were 24 C57/BL6 male mice weighing 25-27g which bought from Pasteur Institute of Iran. Animals were kept in a temperature-controlled room at a 12:12 light/dark cycle with free access to foods and tap water.  Then, they were randomly divided into four groups of 6): Control group (receiving normal diet plus normal saline daily (i.p) for 8 weeks), RA group (receiving normal diet plus 10-mg/kg RA (i.p) daily for 8 weeks), NASH group (receiving Methionine/Choline-Deficient (MCD) diet for 8 weeks to induce NASH), and NASH + RA group (receiving MCD diet plus 10-mg/kg RA for 8 weeks). After 8 weeks, the animals were anaesthetized with a ketamine-xylazine solution and then sacrificed. The abdomen was opened by a midline incision and the liver was immediately removed, weighted and washed in an ice-cold physiological saline. A part of the liver was dissected and kept at –80°C.
A part of the middle lobe was dissected and fixed in 10% natural buffered formalin. Then, 5-μm slices from paraffin-embedded liver tissue were obtained for Hematoxylin And Eosin (H&E) staining. An pathologist blind to the experiment performed histopathological study to determine hepatic steatosis, hepatocyte ballooning, and lobular inflammation.
Results
Total RNA was extracted from the frozen tissue samples using Trizol solution. Complementary DNAs (cDNA) were made from mRNA templates for qualitative real-time PCR. For ELISA assays, a part of the liver tissue was cut and weighted, mixed with Phosphate-Buffered Saline (PBS) buffer, homogenized by an electrical homogenizer, and centrifuged (4000-6000 rpm for 10 min). The prepared supernatants were then assessed for PGE2 levels using an ELISA kit according to the instructions of the manufacturer and read by ELISA reader in 450 nm. Data normality was checked by the Kolmogorov–Smirnov test. Data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variances (ANOVA) was used to compare with the study groups followed by Tukey’s post hoc test in SPSS. P<0.05 was statistically significant.
Discussion
NASH caused excessive lipid droplet accumulation, hepatocyte ballooning, and lobar inflammation in mice. However, RA improved steatosis and edema and reduced inflammation. MMP-9 gene expression significantly increased in the NASH group compared to the control group, while administration of RA significantly reduced the MMP-9 gene expression in the NASH+RA group compared to the NASH group. COX-2 expression significantly increased in the NASH group compared to the control group, but significantly decreased in the NASH+RA group compared to the NASH group. PGE2 level significantly increased in the NASH group compared to the control group, but RA significantly reduced its level in the NASH+RA group compared to the NASH group.


Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Qom University of Medical Sciences (Code: IR.MUQ.REC. 1400.003).
Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.
Authors contributions
All authors contributed equally in preparing all parts of the research.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank Qom University of Medical Sciences for getting ethical code.