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Komeili-Movahhed T, Heidari F, Moslehi A. Evaluation of Rosmarinic Acid Attenuation Effect on Endoplasmic Reticulum Stress in Swiss-type Mice Affected With Nonalcoholic Steatohepatiti. Qom Univ Med Sci J 2023; 17 : 65.8
URL: http://journal.muq.ac.ir/article-1-3657-en.html
URL: http://journal.muq.ac.ir/article-1-3657-en.html
1- Cellular & Molecular Research Center, Qom University of Medical Sciences, Qom, Iran.
2- Cellular & Molecular Research Center, Qom University of Medical Sciences, Qom, Iran. ,moslehi2000@gmail.com
2- Cellular & Molecular Research Center, Qom University of Medical Sciences, Qom, Iran. ,
Keywords: Nonalcoholic fatty liver disease, Endoplasmic reticulum stress, Rosmarinic acid, GRP78, IRE-1
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Introduction
Nonalcoholic steatohepatitis (NASH) is the most common liver disease, characterized by excess lipid accumulation (steatosis) and inflammation in the liver. It has been shown that almost 20% of nonalcoholic fatty liver disease (NAFLD) cases progress to NASH. It is predicted that NASH will become the first indication for liver transplantation in 2020-2025. NASH is characterized by steatosis and inflammation in the hepatocytes, which can progress to cirrhosis, fibrosis, and hepatocellular carcinoma. In NASH pathophysiology, endoplasmic reticulum (ER) stress is a crucial factor; studies have demonstrated that ER stress is one of the principal reasons for developing and promoting this disease through different pathways. Actually, lipid accumulation triggers a cascade of events, such as ER stress, resulting in the destruction and dysfunction of the liver. ER stress creates certain principal markers, including glucose-regulated protein 78 (GRP78), activating transcription factor 6 (AFT6), PKR-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 (IRE1). ER stress results in the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen. These proteins can produce and activate inflammatory cytokines and apoptotic enzymes. ER stress also provokes hepatic stellate cells, Kupffer cells, and other inflammatory cells to produce inflammatory cytokines; cross-reaction between ER stress and inflammation has been demonstrated in many pathological conditions.
Rosmarinic acid (RA) is a synthetic compound that is plentiful in plants from the Lamiaceae family, including Ros-marinus officinalis (R. officinalis) (rosemary), Coleus aromaticus Origanum vulgare L. (oregano), and Thymus vulgaris L. (thyme). Positive effects of RA on diabetes, sepsis, hepatotoxicity, and brain injuries have previously been demonstrated. Rosmarinic acid decreases blood glucose, inhibits insulin resistance, and increases glucose transporter 4 (GLUT4) expression in the skeletal muscles. Studies have shown that Ra can reduce oxidative stress in most tissues and alleviate apoptosis and fibrosis in the renal cells, cardiomyocytes, and hepatic cells. Moreover, it has been shown that administering RA could improve liver steatosis and lipid metabolism-related genes in NASH-induced mice.
As mentioned above, this study aimed to evaluate the lessening effect of rosmarinic acid on ER stress induced by NASH in male mice.
Methods
This study was done at Qom University of Medical Sciences on 24 C57/BL6 male mice (Patur Institute, Tehran, Iran). The animals were weighing 25-27 g. Mice were housed in a temperature-controlled room at a 12:12 light/dark cycle with free access to food and tap water. The animal care and experimental procedure were approved in accordance with the guidelines for animal care and use at Qom University of Medical Sciences. All animals were randomly divided into 4 equal groups (n=6 for each group): Saline group with access to standard diet for 8 weeks and receiving daily saline (IP) simultaneously; rosmarinic acid (RA) group with access to standard diet for 8 weeks and receiving RA (10 mg/kg, IP) for 8 weeks daily; nonalcoholic steatohepatitis group, who received methionine-choline deficient (MCD) diet for 8 weeks to induce NASH; and nonalcoholic steatohepatitis-RA group, who received MCD diet for 8 weeks to induce NASH and rosmarinic acid (10 mg/kg, IP) for 8 weeks simultaneously. After this period, the animals were anesthetized with ketamine-xylazine and killed. Then, midline incisions were done on their abdomens, and their livers were immediately removed, weighed, and washed in ice-cold physiological saline. A part of the liver was dissected and kept at –80°C for real-time assay.
Total RNA was extracted from frozen tissue samples using TRIzol, and the quantity and purity of the RNA samples were measured by a NanoDrop spectrophotometer. Complementary DNAs (cDNA) were made from mRNA templates for qRT-PCR (quantitative reverse transcription- polymerase chain reaction). cDNAs were then used as templates for real-time analysis. Data normality was checked by the Kolmogorov–Smirnov test. The obtained data were expressed as Mean±SEM. Statistical analysis was performed by one-way analysis of variance (to compare the groups) and Tukey's post hoc test using SPSS software. P <0.05 were considered to be statistically significant.
Results
This study showed that GRP78 gene expression significantly increased in the nonalcoholic steatohepatitis group and markedly decreased in the nonalcoholic steatohepatitis-RA group. The mRNA expression of PERK was also significantly upregulated in the nonalcoholic steatohepatitis group, and rosmarinic acid could significantly downregulate PERK expression. In the same way, gene expression of IRE1 showed a marked increase in the nonalcoholic steatohepatitis group; meanwhile, its expression was remarkably lowered in the nonalcoholic steatohepatitis-RA group. However, Apo B gene expression was markedly reduced in the nonalcoholic steatohepatitis group, and its expression was raised in the nonalcoholic steatohepatitis-RA group.
Conclusion
In nonalcoholic steatohepatitis disease, GRP78 is upregulated and induces IRE1 and PERK as ER stress transmembrane sensors in the cytosol. Then, they activate intracellular pathways, such as inflammation and lipogenesis. They also decline lipid excretion from the liver through decreased synthesis of Apo B and keep lipids in the hepatic cells. The findings of this study showed that rosmarinic acid can reverse the expression of these genes and improve nonalcoholic steatohepatitis via ER stress attenuation and increased lipid export from the liver.
Ethical Considerations
Compliance with ethical guidelines
This study approved the Ethics Committee of Qom University of Medical Sciences (Code: IR.MUQ.REC. 1400.003).
Funding
This study was financially supported by Qom University of Medical Sciences.
Authors contributions
All authors contributed equally to this study.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors would like to thank Qom University of Medical Sciences for supporting this study.
Nonalcoholic steatohepatitis (NASH) is the most common liver disease, characterized by excess lipid accumulation (steatosis) and inflammation in the liver. It has been shown that almost 20% of nonalcoholic fatty liver disease (NAFLD) cases progress to NASH. It is predicted that NASH will become the first indication for liver transplantation in 2020-2025. NASH is characterized by steatosis and inflammation in the hepatocytes, which can progress to cirrhosis, fibrosis, and hepatocellular carcinoma. In NASH pathophysiology, endoplasmic reticulum (ER) stress is a crucial factor; studies have demonstrated that ER stress is one of the principal reasons for developing and promoting this disease through different pathways. Actually, lipid accumulation triggers a cascade of events, such as ER stress, resulting in the destruction and dysfunction of the liver. ER stress creates certain principal markers, including glucose-regulated protein 78 (GRP78), activating transcription factor 6 (AFT6), PKR-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 (IRE1). ER stress results in the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen. These proteins can produce and activate inflammatory cytokines and apoptotic enzymes. ER stress also provokes hepatic stellate cells, Kupffer cells, and other inflammatory cells to produce inflammatory cytokines; cross-reaction between ER stress and inflammation has been demonstrated in many pathological conditions.
Rosmarinic acid (RA) is a synthetic compound that is plentiful in plants from the Lamiaceae family, including Ros-marinus officinalis (R. officinalis) (rosemary), Coleus aromaticus Origanum vulgare L. (oregano), and Thymus vulgaris L. (thyme). Positive effects of RA on diabetes, sepsis, hepatotoxicity, and brain injuries have previously been demonstrated. Rosmarinic acid decreases blood glucose, inhibits insulin resistance, and increases glucose transporter 4 (GLUT4) expression in the skeletal muscles. Studies have shown that Ra can reduce oxidative stress in most tissues and alleviate apoptosis and fibrosis in the renal cells, cardiomyocytes, and hepatic cells. Moreover, it has been shown that administering RA could improve liver steatosis and lipid metabolism-related genes in NASH-induced mice.
As mentioned above, this study aimed to evaluate the lessening effect of rosmarinic acid on ER stress induced by NASH in male mice.
Methods
This study was done at Qom University of Medical Sciences on 24 C57/BL6 male mice (Patur Institute, Tehran, Iran). The animals were weighing 25-27 g. Mice were housed in a temperature-controlled room at a 12:12 light/dark cycle with free access to food and tap water. The animal care and experimental procedure were approved in accordance with the guidelines for animal care and use at Qom University of Medical Sciences. All animals were randomly divided into 4 equal groups (n=6 for each group): Saline group with access to standard diet for 8 weeks and receiving daily saline (IP) simultaneously; rosmarinic acid (RA) group with access to standard diet for 8 weeks and receiving RA (10 mg/kg, IP) for 8 weeks daily; nonalcoholic steatohepatitis group, who received methionine-choline deficient (MCD) diet for 8 weeks to induce NASH; and nonalcoholic steatohepatitis-RA group, who received MCD diet for 8 weeks to induce NASH and rosmarinic acid (10 mg/kg, IP) for 8 weeks simultaneously. After this period, the animals were anesthetized with ketamine-xylazine and killed. Then, midline incisions were done on their abdomens, and their livers were immediately removed, weighed, and washed in ice-cold physiological saline. A part of the liver was dissected and kept at –80°C for real-time assay.
Total RNA was extracted from frozen tissue samples using TRIzol, and the quantity and purity of the RNA samples were measured by a NanoDrop spectrophotometer. Complementary DNAs (cDNA) were made from mRNA templates for qRT-PCR (quantitative reverse transcription- polymerase chain reaction). cDNAs were then used as templates for real-time analysis. Data normality was checked by the Kolmogorov–Smirnov test. The obtained data were expressed as Mean±SEM. Statistical analysis was performed by one-way analysis of variance (to compare the groups) and Tukey's post hoc test using SPSS software. P <0.05 were considered to be statistically significant.
Results
This study showed that GRP78 gene expression significantly increased in the nonalcoholic steatohepatitis group and markedly decreased in the nonalcoholic steatohepatitis-RA group. The mRNA expression of PERK was also significantly upregulated in the nonalcoholic steatohepatitis group, and rosmarinic acid could significantly downregulate PERK expression. In the same way, gene expression of IRE1 showed a marked increase in the nonalcoholic steatohepatitis group; meanwhile, its expression was remarkably lowered in the nonalcoholic steatohepatitis-RA group. However, Apo B gene expression was markedly reduced in the nonalcoholic steatohepatitis group, and its expression was raised in the nonalcoholic steatohepatitis-RA group.
Conclusion
In nonalcoholic steatohepatitis disease, GRP78 is upregulated and induces IRE1 and PERK as ER stress transmembrane sensors in the cytosol. Then, they activate intracellular pathways, such as inflammation and lipogenesis. They also decline lipid excretion from the liver through decreased synthesis of Apo B and keep lipids in the hepatic cells. The findings of this study showed that rosmarinic acid can reverse the expression of these genes and improve nonalcoholic steatohepatitis via ER stress attenuation and increased lipid export from the liver.
Ethical Considerations
Compliance with ethical guidelines
This study approved the Ethics Committee of Qom University of Medical Sciences (Code: IR.MUQ.REC. 1400.003).
Funding
This study was financially supported by Qom University of Medical Sciences.
Authors contributions
All authors contributed equally to this study.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors would like to thank Qom University of Medical Sciences for supporting this study.
Type of Study: Original Article |
Subject:
فیزیولوژی
Received: 2023/01/24 | Accepted: 2023/04/26 | Published: 2023/08/1
Received: 2023/01/24 | Accepted: 2023/04/26 | Published: 2023/08/1
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