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. 2020 Jun 12;15(6):e0233767.
doi: 10.1371/journal.pone.0233767. eCollection 2020.

Molecular mechanisms of splenectomy-induced hepatocyte proliferation

Andrey V Elchaninov  1   2 , Timur Kh Fatkhudinov  2   3 , Polina A Vishnyakova  1 , Maria P Nikitina  3 , Anastasiya V Lokhonina  1 , Andrey V Makarov  1   4 , Irina V Arutyunyan  1 , Evgeniya Y Kananykhina  3 , Anastasiya S Poltavets  1 , Kirill R Butov  4 , Igor I Baranov  1 , Dmitry V Goldshtein  5 , Galina B Bolshakova  3 , Valeria V Glinkina  4 , Gennady T Sukhikh  1
Affiliations

Molecular mechanisms of splenectomy-induced hepatocyte proliferation

Andrey V Elchaninov et al. PLoS One. .

Abstract

Functional and anatomical connection between the liver and the spleen is most clearly manifested in various pathological conditions of the liver (cirrhosis, hepatitis). The mechanisms of the interaction between the two organs are still poorly understood, as there have been practically no studies on the influence exerted by the spleen on the normal liver. Mature male Sprague-Dawley rats of 250-260 g body weight, 3 months old, were splenectomized. The highest numbers of Ki67+ hepatocytes in the liver of splenectomized rats were observed at 24 h after the surgery, simultaneously with the highest index of Ki67-positive hepatocytes. After surgical removal of the spleen, expression of certain genes in the liver tissues increased. A number of genes were upregulated in the liver at a single time point of 24 h, including Ccne1, Egf, Tnfa, Il6, Hgf, Met, Tgfb1r2 and Nos2. The expression of Ccnd1, Tgfb1, Tgfb1r1 and Il10 in the liver was upregulated over the course of 3 days after splenectomy. Monitoring of the liver macrophage populations in splenectomized animals revealed a statistically significant increase in the proportion of CD68-positive cells in the liver (as compared with sham-operated controls) detectable at 24 h and 48 h after the surgery. The difference in the liver content of CD68-positive cells between splenectomized and sham-operated animals evened out by day 3 after the surgery. No alterations in the liver content of CD163-positive cells were observed in the experiments. A decrease in the proportion of CD206-positive liver macrophages was observed at 48 h after splenectomy. The splenectomy-induced hepatocyte proliferation is described by us for the first time. Mechanistically, the effect is apparently induced by the removal of spleen as a major source of Tgfb1 (hepatocyte growth inhibitor) and subsequently supported by activation of proliferation factor-encoding genes in the liver.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Dynamics of the liver-to-body weight ratio after splenectomy.
SO, sham operated animals. The data are shown as mean values ± SD with asterisks indicating statistical significance of the differences (as compared with the sham operated control; p < 0.05).
Fig 2
Fig 2
Hepatocyte proliferation in the intact liver after splenectomy: (A) Mitotic figures in hepatocytes, hematoxylin-eosin staining (H&E) and Ki67 expression (FITC, nuclei are counterstained DAPI). Control—sham operated animals; Bars, 50 μm;. Arrows indicate mitotic figures in hepatocytes; Arrowheads indicate Ki67+ cells, (B) Expression of Ccnd1 and Ccne1 in the liver after splenectomy, (C) Content of Ki67, cyclin A, cyclin D1 and cyclin E1 proteins in the liver after splenectomy.
Fig 3
Fig 3. Splenectomy effects on cell proliferation in the liver.
Cryosections of liver tissue after double anti-Ki67 (FITC) and anti-CK18 (PE) or anti-NHF4a (PE) immunostaining. The nuclei are counterstained with DAPI. Bars, 50 μm. Arrows indicate Ki67+hepatocytes, arrowheads indicate non-parenchymal Ki67+ cells.
Fig 4
Fig 4
Profiles of gene (A) and protein expression (B,C) in the spleen. Visual assessment of the western blot was followed by quantitative densitometry. The data are represented as mean values ± SD.
Fig 5
Fig 5. Splenectomy-associated alterations in gene expression profiles of the liver.
EXP, splenectomized animals; SO, sham-operated animals. The data are shown as mean values ± SD with asterisks indicating statistical significance of the differences (as compared with the sham operated control; p < 0.05).
Fig 6
Fig 6. Splenectomy-associated alterations in protein expression profiles of the liver.
EXP, splenectomized animals; SO, sham-operated animals. Visual assessment of the western blot was followed by quantitative densitometry. The data are shown as mean values ± SD with asterisks indicating statistical significance of the differences (as compared to the sham operated control; p < 0.05).
Fig 7
Fig 7. Splenectomy-associated alterations in the representation of different macrophage populations of the liver, as revealed by immunostaining for the macrophage markers CD68, CD163, and CD206.
The diagrams show dynamic changes in the content of CD68+ (IndexCD68), CD163+ (IndexCD163) and CD206+ (IndexCD206) cells after splenectomy. EXP, splenectomized animals; SO, sham-operated animals. The data are shown as mean values ± SD with asterisks indicating statistical significance of the differences (as compared with the sham operated control; p <  0.05).
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Grants and funding

The work of AVE, TKF, PAV, MPN, AVL, AVM, IVA, EYK was supported by the Russian Science Foundation (https://rscf.ru/en/, grant number 17-15-01419). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.