Ann Surg Treat Res.  2020 Jul;99(1):26-36. 10.4174/astr.2020.99.1.26.

Serum level of visfatin can reflect the severity of inflammation in patients with acute cholecystitis

• Park JW
• Kim OH
• Lee SC
• Kim KH
• Hong HE
• Seo H
• Choi HJ
• Kim SJ

• KMID: 2503457
• DOI: http://doi.org/10.4174/astr.2020.99.1.26

Abstract

Purpose
Visfatin is a key cytokine released from the pe ripheral blood mononuclear cells (PBMCs) as well as adipose tissue, and it is involved in immune response as well as inflammation. In this study, we investigated whether the serum visfatin level could be a prognostic factor for predicting the severity of inflammation in patients with acute cholecystitis.
Methods
We examined the blood samples and gallbladder specimens from patients who underwent laparoscopic cholecystectomy for either acute (n = 18) or chronic cholecystitis (n = 18). We determined the visfatin levels of these samples using various procedures such as real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry.
Results
The patients with acute cholecystitis exhibited higher mRNA expression of visfatin in PBMCs, higher serum levels of visfatin, and increased protein expression of visfatin in the gallbladder specimens than in patients with chronic cholecystitis. In the in vitro model of acute cholecystitis, the mRNA expression of visfatin showed the fastest increase among the other pro-inflammatory mediators studied, including interleukin (IL)-10, tumor necrosis factor-, IL-6, intracellular adhesion molecule-1, and ascular cell adhesion molecule-1. Inhibition of visfatin using siRNA abrogated the inhibitory effects of lipopolysaccharide (LPS) on the expression of ABCG1 in GBECs, suggesting that visfatin is significantly involved in the LPS-driven suppression of ABCG1.
Conclusion
Taken together, we concluded that visfatin is a pro-inflammatory mediators that is upregulated during acute cholecystitis and is expected to be increased within a short time after inflammation. Therefore, measuring the serum level of visfatin would be helpful in predicting the inflammatory severity in the patients with acute cholecystitis.

Keyword

ABCG1; Acute cholecystitis; Gallbladder; Inflammation; Visfatin

Figure

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  Fig. 1 Comparison of the expression levels of pro-inflammatory mediators in the blood samples. (A) Real-time polymerase chain reaction showing the differences between the mRNA expression levels of pro-inflammatory mediators in the peripheral blood mononuclear cells (PBMCs) of the patients with chronic and acute cholecystitis. The mRNA levels of the pro-inflammatory markers were significantly higher in the PBMCs of patients with acute cholecystitis than in the chronic cholecystitis patients. Out of all the markers, resistin and visfatin showed the higher increase compared to the expression levels of ICAM-1 and VCAM-1. (B) Enzyme-linked immunosorbent assay results demonstrating the serum levels of pro-inflammatory mediators. The serum levels of the pro-inflammatory markers were significantly higher in acute cholecystitis patients with in chronic cholecystitis patients. Serum levels of visfatin were significantly higher than those of other pro-inflammatory mediators (P < 0.05). Values are presented as mean ± standard deviation of 3 independent experiments. ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; IL, interleukin. *P < 0.05.

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  Fig. 2 Pro-inflammatory mediators expressed in the gallbladder specimens. We obtained 2 areas of grossly inflamed and noninflamed gallbladder tissues, respectively, from patients with acute cholecystitis and chronic cholecystitis. (A) In the gallbladder tissues with acute cholecystitis, the expression levels of visfatin and F4/80 were significantly higher in grossly inflamed tissues than in grossly non-inflamed tissues. (B) Relative densities of pro-inflammatory markers in each group. Values are presented as mean ± standard deviation of 3 independent experiments. *P < 0.05.

• 

  Fig. 3 Comparison of immunohistochemistry of the gallbladder specimens with or without acute cholecystitis. Immunohistochemical staining showed that immunoreactive areas for pro-inflammatory mediators, including visfatin, CD68, ICAM-1, and VCAM-1, were significantly higher in the gallbladder specimens, especially grossly inflamed tissues, of the patients with acute cholecystitis (A) than those of the patients with chronic cholecystitis (B). The percentages of immunoreactive areas were measured using the ImageJ software (National Institute Health, Bethesda, MD, USA) and were expressed as values relative to those in normal gallbladder. Values are presented as mean ± standard deviation of 3 independent experiments. ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1. *P < 0.05.

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  Fig. 4 Changes of mRNA expression of pro-inflammatory mediators over time in the in vitro model of acute cholecystitis. (A) Determination of LPS concentration for generating in vivo model of acute cholecystitis. The most pro-inflammatory mediators significantly increased when the LPS of concentration above 25-ng/mL concentration was added. We thus use the 25-ng/mL LPS to generate the in vitro model of acute cholecystitis. (B) Real-time polymerase chain reaction showing the time-dependent differences in the levels of these pro-inflammatory mediators in the in vitro model of cholecystitis. The mRNA expression of visfatin was increased in the shortest time span compared to those of the other pro-inflammatory mediators. Values are presented as mean ± standard deviation of 3 independent experiments. ICAM-1, intracellular adhesion molecule-1; LPS, lypopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1. *P < 0.05.

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  Fig. 5 Determination of the role of visfatin during acute cholecystitis. (A) Real-time polymerase chain reaction comparing the mRNA expression of the pro-inflammatory mediators, including TNF-α, IL-10, and ICAM-1, before after blocking visfatin expression with siRNA. When gallbladder epithelial cells (GBECs) were treated with LPS, these pro-inflammatory mediators were significantly increased (P < 0.05). However, blocking the expression of visfatin with siRNA significantly reduced the expression of these pro-inflammatory mediators. (B) Western blot analysis comparing the expression of ABCG1 before and after blocking visfatin expression with siRNA. When visfatin was not inhibited, GBECs treated with LPS significantly inhibited the expression of ABCG1. However, inhibition of visfatin by si-visfatin significantly abrogated the suppressive effects of LPS on the expression of ABCG1 in the GBECs. Values are presented as mean ± standard deviation of 3 independent experiments. Ct, control; siCt, negative control siRNA; ICAM-1, intracellular adhesion molecule-1; LPS, lypopolysaccharide; SRA, scavenger receptor A; TNF-α, tumor necrosis factor-α; IL, interleukin. *P < 0.05.

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