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Anna Khomenko, O. Lisakovska, M. Veliky
e-mail: annavic@ukr.net
Laboratory of Medical Biochemistry, Palladin Institute of Biochemistry of NASU, str. Leontovich 9, Kyiv, Ukraine, 01601
Background. Glucocorticoid (GC) therapy associated side effects could be ascribed to inhibition of vitamin D3 metabolism. Changes in cholecalciferol hydroxylation to
25OHD3 can be linked to prednisolone induced dysfunction of hepatocytes. Therefore, the idea of this work is to substantiating the ability of vitamin D3 to correct im-
pairments of the functional activity of hepatocytes caused by chronic administration of synthetic glucocorticoid – prednisolone (P).
Conclusions. In summary, prednisolone-induced impairments of the functional activity of hepatocytes are largely mediated by the development
of D-deficiency and can be effectively influenced by administration of vitamin D3.
Cytology of isolated hepatocytes
(staining by Romanovsky-Gimza and H&E)
1
Expression of Bax and Bcl-2 in hepatocytes
(by immunocytochemistry)
Groups
(n=15)
25ОНD3, nmol/l
of blood serum
(by ELISA)
1 - Intact rats 110,0±4,7
2 - 0,5 mg P 31,2±0,9*
3 - 0,5 mg P + 100 IU D3 89,3±2,0#
It was shown that prolonged prednisolone administration at dose 5 mg per kg of b.w. per os (daily for 30 days) lowered the level of 25OHD3 (by 70%) in serum and inhibited
two-fold the total activity of vitamin D3 25-hydroxylase in hepatocytes vs. control. Prednisolone reduced the content of CYP27A1 and CYP2R1 isoforms of 25-hydroxylase.
Vitamin D3 (100 IU) administration resulted in normalization of vitamin D3 metabolism.
3
2
Bax
Bcl-2
1 2 3
Prednisolone-induced cellular damage was further supported by a significant increase in
the number of hepatocytes capable to accumulate PI that is associated with necrotic cell
death. Simultaneously observed increase in the expression of antiapoptotic protein Bcl-2
indicates activation of antiapoptotic signaling in hepatocytes.
The positive effect of vitamin D3 in the normalization of disturbances caused by predniso-
lone action comprised decreasing degenerative disorders in hepatocytes and enhancing
proapoptotic activity, decreased formation of RNS, lower nitrative protein modification and
expression of iNOS.
The percentage of necrotic death of hepatocytes
(by flow cytometry with propidium iodide)
Total activity of vitamin D3 25-hydroxylase in isolated hepatocytes
(by radioreceptor assay using [3Н]-25ОНD3)
1 2 3
The nitric oxide formation in isolated
hepatocytes:
A - representative histograms of DAF
fluorescence; B - quantification of
DAF oxidation documented by flow
cytometry analysis.
Levels of iNOS (A) and nitrated proteins (B)
in liver tissue (by Western blot)
* - vs intact rats, p<0.05; # - vs prednisolone rats, p<0.05
Groups
(n=15)
Hepatocytes
per 1g of liver,
n∙106
Intact 22,0 ± 0,5
0,5 mg P 11,0 ± 0,3*
0,5 mg P + 100 IU D3 18,7 ± 0,4#
FUNCTIONAL ACTIVITY OF HEPATOCYTES:
EFFECTS OF PREDNISOLONE AND VITAMIN D3
CYP27A1, 60 kDa
β – actin
β – actin
CYP2R1, 57 kDa
1 2
Levels of CYP27A1 and CYP2R1 proteins
in liver tissue (by Western blot)
A
B
Palladin Institute of Biochemistry
of the National Academy of Sciences of Ukraine
* - vs intact rats, p<0.05; # - vs prednisolone rats, p<0.05
* - vs intact rats, p<0.05; # - vs prednisolone rats, p<0.05
Relative fluorescence (fold)
Increased generation of RNS and the expression of iNOS, nitrated proteins related to prednisolone
action caused disruption of the integrity of hepatocytes and thus reducing the number of functional-
ly active hepatocytes.
It was found prednisolone caused the in-
crease in the number of hepatocytes with the
signs of destructive changes in the cytoplasm
and nuclei.
A B
0
2
4
6
8
10
12
1
pmol 25OHD3/hour/106
hepatocytes
2
3
#
*
0
2
4
6
8
10
12
14
16
1 2 3
% of the total number
of events
*
#
* - vs intact rats, p<0.05; # - vs prednisolone rats, p<0.05