E-ISSN : 2757-7163
Materials and Methods: Twenty-four Wistar rats were used for the experimental study. Each group comprised of 8 subjects. Sham group (Group 1) was nephrectomized for histopathologic examination and blood samples were obtained for biochemical analysis. Control group (Group 2) was subjected to ischemia for 1 hour and reperfusion for 24 hours, then they were nephrectomized for histopathologic examination and blood samples were taken for biochemical analysis. Treatment group (Group 3) was given 80 mg/kg diosminhesperidin combination (DAFLON®) for 10 days and subjected to ischemia for 1-hour and reperfusion for 24 hours, then they were nephrectomized for histopathologic examination and blood samples were obtained for biochemical analysis.
Results: As a result of biochemical analysis and histopathologic examination, more significant results were acquired in regard to other groups. Serum urea values were statistically significant (p<0.05). Consistent with the results of the studies previously performed, higher creatinine values were detected in the control and treatment groups. Enzymatic activities of superoxide dismutase and glutathione peroxidase were significantly lower in the treatment group when compared with the control group. When groups were compared based on histopathologic examination findings, cell necrosis and ischemic alterations were statistically significant parameters (p<0.05). When all parameters were analysed, values indicating any histopathologic abnormalities were at the lowest level in the treatment group. Histopathologic alterations were most frequently detected in the control group.
Conclusion: Anti-inflammatory activity of diosmin-hesperidin which is used experimentally in the treatment of ischemia-reperfusion injury was evaluated both biochemically and histopathologically. Based on the literature data, it is thought that the damage occurring after ischemia-reperfusion injury can be prevented with diosmin-hesperidin treatment.
The response that organs give to experimental ischemiareperfusion injury was very well specified in the rats and rats were preferred as experimental animals in most of the literature studies [2-6]. During ischemia, the tissue is damaged by asphyxiation and when the normal blood flow is retrieved, tissue damage aggravates greatly as a result of a series of events caused by the oxygen entering the atmosphere [7,8].
Mitochondrial electron transport chain reactions, inhibition of arachidonic acid metabolites, increase in intracellular calcium levels, xanthine oxidase system, iron ion, etc. involve in the production of FORs which induce ischemia-reperfusion injury in the kidney tissue. These factors affect each other sequentially and disrupt cell functions, increase membrane destruction, and result in the production of endogenous toxins [9-13].
The only way of treatment in ischemic kidneys is to increase the renal blood flow through reperfusion. Yet in that case reperfusion damage inevitably occurs. In such a case, since the increase of blood supply is inevitable, it is necessary to look for solutions to prevent reperfusion damage. Many agents such as vitamin E, melatonin, phospholipase type 3 enzyme inhibitors (amrinone, olprinone), adenosine, n-acetyl cysteine, nitric oxide (NO), calcium channel blockers, mycophenolate mofetil have been used in order to prevent or alleviate renal ischemia-reperfusion injury associated with various etiologic factors [10,11].
Diosmin-hesperidin is produced by the purification of flavonoid extracts of a plant found in the nature. Daflon is a phlebotonic and vasculoprotector agent that is comprised of 90% diosmin and 10% hesperidin. Hesperidin reinforces the activity of diosmin, improves wound recovery by acting against inflammatory mediators and protects microcirculation via decreasing blood viscosity [14].
Diosmin-hesperidin has also shown anti-inflammatory effects through many mechanisms of action in our study. Many studies have been conducted on significant ischemia-reperfusion injury preventing effects of diosmin-hesperidin in multiple organs such as heart, brain muscle tissue, and peritoneum [14,15].
Diosmin-hesperidin is an important antioxidant drug combination. Leukocyte aggregation is important in ischemiareperfusion injury and damage can be prevented with diosminhesperidin at daily oral doses of 500 mg. Also, this drug combination reduces the amount of H2O2 released from leukocytes by suppressing activity of myeloperoxidase (MPO). A decrease in the MPO activity can explain the decrease in H2O2 in the group that received diosmin-hesperidin at doses protecting against oxidative stress associated with glutathione (GSH). Diosmin-hesperidin given at doses protecting against oxidative stress associated with GSH guards the escape of macromolecules from the microvascular structures. According to histopathological data, diosmin-hesperidin prevents infiltration of leukocytes into the perivascular area. Although it does not totally prevent leukocyte infiltration, a significant reduction in leukocyte accumulation in the perivascular area in the kidney was observed [16].
We have aimed to experimentally investigate the activity of diosmin-hesperidin, which acts against inflammatory mediators in ischemia-reperfusion injury and protects microcirculation by decreasing blood viscosity in cases with renal ischemiareperfusion injury.
In this study, rats were separated into three groups of 8 subjects. Experimental animals were tracked for 10 days by being kept at normal room temperature and in separate cages during preoperative and postoperative periods. Groups 1 and 2 were followed up without giving any treatment. Daily doses of 80 mg/kg diosmin-hesperidin (DAFLON®) were given to Group 3 subjects for 10 days by crushing film-coated tablets and adding them in their drinking water. At the end of the 10th day, only Group 1 rats underwent nephrectomy through laparotomy incision. In Groups 2 and 3, ischemia was induced in the renal artery for 60 minutes. After 24 hours, for the subjects in each group, before the procedure, serum samples were collected for the measurement of glutathione peroxidase, superoxide dismutase and urea creatinine. Then nephrectomy specimens in 10% formaldehyde solution were sent for histopathologic evaluation (Figure 1).
After tissue samples in 10% formaldehyde were embedded in paraffin blocks and stained with hematoxylin-eosin, all preparations were examined under light microscope by a pathologist blinded to the study protocol. A semiquantitative scale defined by Paller et al. who evaluated, and scored the alterations in acute renal insufficiency was used [1].
Modified scoring system defined by Paller et al:
1- Chronic inflammation (0, +, ++)
2- Tubular epithelial cell flattening (0, +, ++)
3- Cytoplasmic vacuoalization (0, +, ++)
4- Cell necrosis and ischemic changes (0, +, ++)
5- Tubular lumen obstruction (0, +, ++)
<
b>Statistical Analysis
Data analysis was performed using the SPSS 15 package
program. While descriptive statistics were shown as mean ±
standard deviation for blood creatinine values, alterations in
histopathologic results and blood parameters were shown as
average (minimum-maximum) values. One-Way Anova analysis
was used in the comparisons of blood parameters both within
and between groups. T-test with Bonferroni correction was used
to investigate whether there is a significant alteration in the
groups in regard to serum urea creatinine levels. Percent changes
between groups in serum urea creatinine levels within 24 hours
were calculated, and intergroup comparisons were performed.
The statistical significance of histopathologic scores between
groups was analyzed using the Kruskal-Wallis test. The results
were accepted as statistically significant at p<0.05.
Figure 2a: Range of urea values between groups 2b. Range of creatinine values between groups
Serum Creatinine Values
Average serum creatinine values were higher in the control
and the treatment groups compared to the sham group. There
was no statistically significant difference between the groups in
this regard (p>0.05) (Figure 2).
Superoxide Dismutase Enzyme Values
Superoxide dismutase enzyme values were higher in the
control group compared to the other groups. The sham group
and treatment group had approximately the same values. There was no statistically significant difference between the groups in
this regard (p>0.05).
Glutathione Peroxidase Enzyme Values
Highest glutathione peroxidase enzyme values were found
in the control group. The sham group and treatment group
had approximately the same values. There was no statistically
significant difference between the groups in this regard (p>0.05).
Results of Histopathological Evaluation
According to histopathological classification defined by
Paller et al., a total of 100 cortical tubules from 10 different
microscopic field of views were evaluated in the kidney
preparation of each experimental animal. An average score for
each case was stated by averaging the scores. From the average
scores of 8 rats in each group, the general average and standard
deviation were calculated.
Histopathological evaluation revealed the presence of significant intergroup differences. Especially cell necrosis, tubular lumen obstruction, chronic inflammation, cytoplasmic vacuolization, and epithelial cell flattening were more common in the control and treatment groups when compared with the sham group. The most distinct histopathological alterations were cell necrosis and inflammation which were observed significantly less frequently in the treatment group relative to the control group (Figure 3).
There were histopathological differences between the control group and the treatment group. Histopathological alterations which are the indicator of kidney insufficiency were more common in the control group. The frequency of histopathological alterations in the treatment group was nearly close to the sham group. The most distinct alteration was cell necrosis and ischemic changes were less common in the treatment group. Cell necrosis and ischemic alterations were statistically significant findings (p<0.05).
Histopathological alterations indicating all reperfusion injuries were observed more commonly in the control group. In the treatment group, severe histopathological alterations were observed less frequently compared to the control group. The histopathological alterations caused by kidney reperfusion injury in the treatment group were observed significantly less frequently compared to the control group. The most distinct alteration was cell necrosis and ischemic changes were less common in the treatment group (p<0.05) (Table 1).
Table 1: Histopathological data
As a result of histopathological evaluations, the most distinct alteration was found in the treatment group. As a critically important finding cell necrosis and ischemic alterations were significantly different between the treatment, and control groups. There were partial cell necrosis and ischemic alterations in the treatment group, while cell necrosis and ischemic alterations were more frequently seen in the control group with a statistically significant intergroup difference (p<0.05) (Figure 3).
In our study; we observed that in the model we formed by applying ischemia for 60 minutes and reperfusion for 24 hours, the levels of plasma urea and creatinine which are indicators of renal glomerular insufficiency in the treatment and the control groups increased. While serum urea values were statistically significantly higher (p<0.05), creatinine values were not. However, their median average values were significantly increased. Scarce number of data, uncertainties about the dosages and time periods specified for the treatment were thought to be the reasons for not obtaining statistically significant results . A significant reduction in activities of renal antioxidant enzymes and histopathological manifestations suggestive of necrosis was observed. Low antioxidant enzyme activity is an indicator of a mild ischemia-reperfusion injury. Enzyme activities were at the highest level in the control group. Because this group was exposed most often to the adverse effects of ischemia-reperfusion injury compared to the other groups. In the sham and treatment groups, enzyme activities were almost at the same level and quite low compared to the control group. As a result of this finding, it can interpreted that the treatment group was least affected by ischemia-reperfusion injury.
When studies were reviewed, there was no specific dosage used for the prevention of renal ischemia-reperfusion injury [1,5]. An average dose used in our study was determined based on the studies conducted on other organs.
Histopathologically there was a statistically significant decrease, especially in cell necrosis and ischemic alterations (p<0.05). The scoring system defined by Paller et al. [1] and used in acute kidney insufficiency was modified and applied in our study. We detected that the treatment group was more effective in improving all parameters taken into consideration and less damaging to the cell relative to the control group. Our findings were consistent with those of many studies investigating therapeutic activity of diosmin-hesperidin in muscle tissue, brain tissue, peritoneum, and heart tissue with ensuing histopathologically significant results [14,15].
Dobashi et al. applied renal ischemia (30, 60, 90 min.) and reperfusion (2, 24, 72, 120 hours) for different time periods, and noticed a significant decrease in superoxide dismutase and glutathione peroxidase activities and increase at the level of lipid peroxidation in the group in which they applied ischemia for 60 minutes and reperfusion for 24 hours [15]. Also in the studies where ischemia and reperfusion were applied for different durations, decrease in glutathione levels whereas increase in peroxidation and plasma urea and creatinine levels were indicated. Our findings were consistent with the results of previous studies [15,17].
Many studies cited in the literature have shown the favborable effect of diosmin-hesperidin on alleviating or preventing ischemia-reperfusion injury [14,15]. In our study, diosminhesperidin at a dose of 500 mg that prevented the development of ischemia-reperfusion injury in other organs was also found to be effective in preventing renal ischemia-reperfusion injury. Diosmin-hesperidin is a medication used in venous insufficiency of extremities and venous system diseases such as hemorrhoids. Its venous tone enhancing, capillary resistance, and capillary permeability regulating, and lymphatic drainage enhancing effects are known. Besides, its free radical scavenging and leukocyte adherence-reducing effects have been shown. Duchene-Marullaz et al. demonstrated that it significantly reduced free radical activity, chemotactic reaction, and free radical activity induced by leucocyte phagocytosis consistent with our findings [18].
Stucker et al. conducted a microvascular permeability study using fibrils of cremaster muscle, and indicated that the treatment with diosmin-hesperidin also significantly reduced the residue induced by ischemia and bradykinin in vascular permeability [19]. In in vitro and in vivo studies, Di Peri and Auteri demonstrated that diosmin-hesperidin reduced the activity of the complement system, and this mechanism of action was considered to constitute one of the anti-inflammatory effects of this drug therapy which also contributes favorably to the ulcer recovery [20].
Schoab et al. conducted a study on patients with chronic venous insufficiency using diosmin-hesperidin, and showed that it increased wound recovery by reducing oscillations in endothelial VCAM1 and ICAM1 expressions, preventing leukocyte adhesion, activation, and migration which consequently shortening the recovery time in patients with venous insufficiency [21]. Coloridge-Smith et al. conducted a meta-analysis on patients with venous ulcers developed as a result of chronic venous insufficiency, and showed that administering diosmin-hesperidin in addition to conventional treatment was effective in the rapid reduction of ulcer-related symptoms and sizes of ulcers compared to conventional treatment only [22].
Hasanoğlu et al., showed that diosmin-hesperidin was systematically and topically effective in infected skin injuries and improved wound recovery [23,24]. Pecking et al. investigated the effects of diosmin-hesperidin in upper extremity lymphedema developed after breast cancer surgery, and showed that diosmin-hesperidin was an effective treatment of lymphedema by increasing lymphatic flow in lymphedema, significantly reducing intralymphatic pressure and at the same time increasing the return of interstitial fluid to the capillaries [16]. Hasanoğlu et al. used diosmin-hesperidin systematically and topically in infected second-degree burns and showed that wound recovery was faster and better in patients in whom diosmin-hesperidin was used [24].
Many above-mentioned studies have indicated that diosminhesperidin was effective in preventing inflammatory effects and injury caused by free oxygen radicals. Therefore, frequent use of diosmin-hesperidin at the experimental level as an antiinflammatory drug in ischemia-reperfusion injury conveys extreme importance as has been always observed in previously performed studies. Our study has shown that diosmin-hesperidin exerted anti-inflammatory effects that prevented development of ischemia-reperfusion injury.
Ethics Committee Approval: Ethics committee approval for the study was acquired from the Ethics Committee of Ankara Training and Research Hospital (Date: 18.02.2009, Decision No: 2347).
Informed Consent: An informed consent was obtained from all the patients.
Publication: The results of the study were not published in full or in part in form of abstracts.
Peer-review: Externally and internally peer-reviewed. Authorship Contributions: Any contribution was not made by any individual not listed as an author. Concept – K.Y., E.E.; Design – K.Y., E.E.; Supervision – K.Y., E.E.; Resources – K.Y., E.E.; Materials – K.Y., E.E.; Data Collection and/or Processing – K.Y., E.E.; Analysis and/or Interpretation – K.Y., E.E.; Literature Search – K.Y., E.E.; Writing Manuscript – K.Y., E.E.; Critical Review – K.Y., E.E.
Conflict of Interest: The authors declare that they have no conflicts of interest.
Financial Disclosure: The authors declare that this study received no financial support.
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