trans -Chalcone protects male rats against high-fat emulsion-induced pancreatic injury by inhibiting TNF-α and MCP-1 expression

Background. High-fat diet (HFD) intake is linked to ectopic fat deposition in the pancreas. It also causes pancreatic inflammatory lesions. trans -Chalcone is a simple chalcone with protective effects against HFD-induced metabolic disorders. This study, for the first time, explored the possible effects of this chalcone on high-fat emulsion-induced pancreatic abnormalities in rats. Methods. Twenty-one male rats were randomly assigned into three groups: control (received 10% tween 80); HFD (received high-fat emulsion+10% tween 80); and HFD + chalcone (received high-fat emulsion+ trans -chalcone). Real-time PCR was used to assess pancreatic mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α) in the pancreas of all rats. Results. High-fat emulsion increased the mRNA expression levels of inflammatory biomarkers, including TNF-α and MCP-1 in the pancreas of rats, and treatment with trans -chalcone prevented these high-fat emulsion-induced changes. Conclusion. trans -Chalcone can protect the pancreas of male rats against HFD-induced abnormalities through its anti-inflammatory effects. Practical Implications. It seems that consumption of HFD up-regulates the production of inflammatory mediators in the pancreas. On the other hand, trans -chalcone can ameliorate HFD-related pancreatic inflammation.

therapeutic and pharmacological activities.trans-Chalcone is a simple chalcone with protective effects against HFD-induced metabolic syndrome, pulmonary inflammation, liver steatosis, and nonalcoholic steatohepatitis (NASH).The present study investigated the possible effects of this chalcone on the expression levels of TNF-α and MCP-1 in the pancreas of high-fat emulsion-fed rats.

Experimental protocol
Twenty-one male Wistar rats (body weight: 210-250 g) were maintained in an environment with controlled conditions (temperature: 22±2 °C, lightdark cycle: 12/12 hours, free access to standard food and water).The animals were randomized into three groups (7 rats per group) as follows: 1) Control group: received 10% Tween 80, 2) HFD group: received high-fat emulsion and 10% Tween 80, and 3) HFD+chalcone group: received high-fat emulsion and trans-chalcone.Animal treatments were started after the acclimatization period (one week).Then, oral gavages of 10% Tween 80 (2 ml), high-fat emulsion (10 ml/kg), and trans-Chalcone (20 mg/kg, dissolved in 10% Tween 80) were carried out for six weeks (once daily).At the end of the treatment periods, overnight fasted (fasting period: 8 h) animals were deeply anesthetized.Then, pancreas and blood samples were collected.The levels of fasting blood glucose were measured utilizing a digital glucometer (Gluco Sure, Star, Taiwan).Pancreas samples were frozen with liquid nitrogen immediately after preparation and kept in a -70°C freezer for further assays.

Real-time PCR
Total RNA from homogenized pancreas samples was extracted by RNX-Plus solution kit (Fermentase, Cinagen Co., Tehran, Iran).Then, the synthesis of complementary DNA (cDNA) was done using Thermo Scientific RevertAid Reverse Transcriptase (Thermo Scientific, Waltham, MA).Real-time PCR by SYBR Green PCR Master Mix was performed to detect mRNA expression levels of TNF-α and MCP-1.Also, β-actin was used as a housekeeping gene.Real-time PCR was carried out in a Rotor-Gene 6000 Real-time PCR machine.Relative mRNA expression levels of TNF-α and MCP-1 were analyzed using 2 −ΔΔCt method.

Statistical analysis
All data were analyzed through GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA).Values were shown as mean ± standard deviation (SD).One-way analysis of variance (ANOVA) followed by Turkey's post hoc test were used.A P-value less than 0.05 was considered significant.

Results
In the present study, treatment with the high-fat emulsion for six weeks significantly (P<0.001)increased fasting blood glucose levels in the HFD group compared with the control group.On the contrary, treatment with trans-chalcone for six weeks significantly (P<0.001)decreased blood glucose levels in the HFD+chalcone group compared with the HFD group.To elucidate whether high-fat emulsion and trans-Chalcone modify the mRNA expression levels of inflammatory mediators, including TNF-α and MCP-1, Real-time PCR assay was performed.The results indicated that mRNA expression levels of TNF-α significantly (P<0.001)increased in the pancreas of animals in the HFD group compared with the control group.On the contrary, treatment with trans-Chalcone significantly inhibited these high-fat emulsion-induced changes in TNF-α mRNA levels.In this case, trans-Chalcone significantly (P<0.05)decreased the expression levels of this pro-inflammatory cytokine in the HFD+chalcone compared with the HFD group.Similarly, high-fat emulsion significantly (P<0.001)increased expression levels of MCP-1 in the pancreas of rats in the HFD group compared with the control group.In contrast, treatment with trans-Chalcone significantly (P<0.001)reduced MCP-1 mRNA levels in the HFD+chalcone group compared with the HFD group.

Conclusion
In the present study, consumption of high-fat emulsion caused pancreas inflammation in male rats, which was reflected by up-regulation of MCP-1 and TNF-α mRNA expression levels.On the other hand, trans-chalcone effectively inhibited these high-fat emulsion-induced pancreatic changes.Rebours et al. proposed that chronic HFD led to pancreatic inflammation and pancreatopathy in rats.It seems that MCP-1 is involved in the inflammation and fibrosis in the pancreas.Up-regulation of this chemokine has been observed during acute and chronic pancreatitis both in animal and human studies.In the pancreas, MCP-1 is secreted in response to pro-inflammatory stimuli such as TNF-α.Besides, it has been suggested that MCP-1 increases adipogenic genes expression and reduces insulinstimulated uptake of glucose.Therefore, the downregulation of MCP-1 could be a key mechanism for the protective role of trans-chalcone against chronic pancreatopathy.Overall, we witnessed the protective effects of trans-chalcone on pancreatic abnormalities in high-fat emulsion-fed animals.