الفهرس 
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Abstract
Diabetic nephropathy refers to the deterioration of kidney function seen in chronic T1DM and T2DM patients. DN is not only the most common cause of end-stage renal disease world-wide but also increases the risk of mortality up to fourteen times compared to normoalbuminuric diabetic patients. DN as a microvascular complication of diabetes is undoubtedly one of the main health concerns facing our society.
The progression of this disease is known to occur in serious of stages and is linked to hyperglycemia. Genetic and environmental factors also play an important role in the progression of DN. While common pathological features of these diseases are glomerular hemodynamic changes, renal structure abnormalities, oxidative stress as a common mediator and disruption of several metabolic pathways have been thought to partly represent the cascades underlying their characteristic epigenetic alternation.
Due to the complexity of these multifactorial disease, current treatment strategies are only useful as symptomatic relief agents for DN and to prevent the progression of diabetes without side effects. Thus, it is necessary to develop safe and more effective therapeutic/preventive strategies to combat these devastating diseases.
Consequently, the use of phytochemicals or herbal medicines in fighting chronic disease, in general, and particularly DN is being thoroughly investigated as they are shown to be protective against DN biomarkers, oxidative stress and inflammation.
Phytochemicals have been consistently proposed as alternative therapy in modern medicine, but their efficacy is somewhat limited by rapid metabolism, insufficient permeability across membranes and decreased its bioavailability and stability in tissues. Fortunately, current advances in nanotechnology present opportunities to overcome such limitations and increased the efficiency and delivery to the target sites by several folds.
The current study aims to examine the possible regulatory effects of QUNPs in compare with native quercetin either with or without metformin against the abnormal molecular and biochemical pathways that eventually culminate in DN hamsters. Also, evaluation of the cytotoxicity of quercetin either in native or nano form in healthy hamsters.
This study was divided into three sections:
The first section included the characterization of quercetin by HR-TEM. HR-TEM analysis showed that the mean diameter of QUNPs was 91 ± 0.8 nm as well as QUNPs had a nano rod and smooth surface.
The second section included evaluation of the cytotoxicity of quercetin either in native or nano form in healthy male adult hamsters. It revealed that the dose of 160 mg/kg body weight considered the LD50 for either native or nano quercetin which recorded 50% death of animals. On the other hand, the oral dose of 40 mg/kg body weight of either native or nano quercetin have no toxic effects which illustrated by the absence of dead animals.
The third section included the animal trial. One hundred and five (105) healthy adult male Syrian hamsters weighing 190 ± 10 g were used after 7 days of acclimatization period and offered the balanced diet with drinking water ad libitum.
* To induce DN, hamsters consumed HF-HFr diet for 5 consecutive weeks after the acclimatization period, and at the beginning of the third week, these animals were injected intraperitoneal with a single dose of STZ (35 mg/kg body weight) by dissolving 1.57 g STZ in 50 ml 0.1 M cold sodium citrate buffer (pH 6.3) for induction of T2DM, and by the end of the fifth week the incidence of DN was screened by measuring blood and urine creatinine concentration from overnight fasted animals (9 hours) as well as urine volume to measured GFR. The results of GFR resulted in significant reduction in the level of blood creatinine with a decrease in the level of creatinine in urine and an increase urine volume, which indicated the occurrence of the disease. And after confirming the incidence of this disease, the animals continued to fed HF-HFr diet until the end of the experiment period.
The animals were divided as the following :
G1: Healthy control,
G2 DN* untreated hamsters,
G3: DN*+ metformin (100 mg/kg body weight/day),
G4: DN*+ native quercetin (20 mg/kg body weight/day),
G5: DN*+ QUNPs (20 mg/kg body weight/day),
G6: DN*+ native quercetin (20 mg/kg body weight/day( + metformin (50 mg/kg body weight/day(
G7: DN*+ QUNPs (20 mg/kg body weight/day(+ metformin (50 mg/kg body weight/day(.
The results illustrated that:
1- The nutritional study data confirmed the DN caused a statistically significant (P<0.05) reduction in the mean values of body weight, FER and FCR in the DN hamsters when compared with the healthy group by about 38.15 %, 59.18 % and 147.89%, respectively. Whereas significant increment in the feed intake and relative weight of kidneys was observed in DN untreated hamsters when compared with the healthy group by 53.5 % and 116.21 %, respectively. While all treatment of DN hamsters with consuming native or nano quercetin with and without metformin oral doses caused significant (P<0.05) improvement in all nutritional evaluation as compared to DN untreated group (G2).
2- Concerning nephropathy biomarkers, the results of GFR showed a significant (P<0.05) decrease in DN untreated group (G2) by 86.18% when compared with their corresponding healthy control hamsters. In contrast, DN hamsters in G7 which consumed oral doses of QUNPs with metformin, showed a significant increment in GFR levels by 291.49% compared to G2. Also, a statistically significant increase in GFR was observed in all treated groups.
3- Regarding IRS-1 and GLUT-4 gene expression, there was a highly significant decrease in the expression of these genes in DN untreated group. Consumption of oral doses of QUNPs with metformin showed an increase in IRS-1 and GLUT-4 gene expression by 618.18% and 2533.33%, respectively as compared to DN untreated group. Results of IRS-1 and GLUT-4 gene expression were significantly confirmed by immunohistochemical analysis, where DN received QUNPs with metformin in G7 achieved a highly IRS-1 and GLUT-4 expression (score 3, +++).
4- Results of epigenetic markers confirmed that induction of DN was accompanied by a state of global histone hyperacetylation. This was instigated by significant elevation in HDAC activity in DN untreated hamsters by 106.8% compared to G1. On the contrary, the combination treatment with QUNPs and metformin were able to significantly inhibit the activity of HDAC when compared to DN untreated group. Therefore, the role of this treatment in orchestrating several biological pathways was suggested to be dependent on epigenetic regulation of DN.
5- Our results also confirmed that diabetic biomarkers correlated with change in IRS-1 and GLUT-4 gene expression. Markedly, QUNPs with metformin treatment in G7 caused improvement in levels of blood glucose (66.27%), insulin (276.19%), HOMA-IR (41.77%), β-cells function (818.45%), HbA1c (67.89%) and C-peptide (256.09%) when compared to G2. Our results revealed that oral treatment with native or nano quercetin with metformin brought back the diabetic biomarkers to near normal levels.
6- Concerning renal function biomarkers, DN untreated group exerted a marked increment in blood levels of urea, uric acid, creatinine, globulin and urine albumin by 366.16%, 283.41%, 764.44, 41.33% and 183.37%, respectively while caused decrement in blood level of total protein, albumin, A/G ratio and urine creatinine by 42.29%, 81.33%, 87.15% and 78.86%, respectively compared to G2. Whereas, there was a significant reduction in serum urea, uric acid, creatinine, globulin and urine albumin in G6 and G7 to near the normal level, the reduction for G6 was by 47.6%, 50.85%, 66.83%, 23.5% and 52.94%, respectively and for G7 was 52.9%, 56.75%, 69.66%, 26.1% and 60.78%, respectively compared to DN untreated group.
7- As for the effect of the tested oral treatments on diabetic nephropathy biomarkers in kidney tissues, DN induction cause significantly (P˂0.05) an elevation in renal AGEs, 8-OHgG, COX-2 activity and AR activity by 184.45%, 362.26%, 117.08 and 174.91%, respectively compared to G2. While QUNPS with metformin surpassed the other tested treatments in their effects on metabolic function as it thought to be the main etiologic factors of the established renal deficits and microvascular damage in kidney tissues.
8- Also, our results confirmed that epigenetic alteration that correlated with defect in renal function was also accompanied by inflammation and DNA fragmentation. The level of proinflammatory cytokines; IL-6 and TNF-α as well as DNA fragmentation percentage were all increased in DN untreated group by 129.87%, 616.7% and 462.74%, respectively as compared to healthy group. While treated groups with oral doses of native or nano quercetin with or without metformin cause diminished in these inflammatory biomarkers. The overwhelming consensus of the superiority of QUNPs + metformin to the other treatments were soundly confirmed by their effects on these markers as well as the percentage of DNA fragmentation.
9- Concerning the antioxidant and oxidative stress biomarkers in kidney tissues, DN untreated group showed a statistically significant (P<0.05) decrease level of renal SOD and GPx activities by 58.61 % and 59.11 %, respectively whereas increase in oxidative stress status presented by increased renal MDA and NO by 342.07 % and 123.38 %, respectively when compared with the healthy group. Concisely, the results indicated increased anti-oxidant status and reduced oxidative stress status with oral consumption of oral doses of native or nano quercetin with and without metformin by abolishing the accumulation of renal MDA and NO, accompanied by increased levels of renal SOD and GPx activities as compared with their corresponding DN untreated group.
10- Collectively, all the previous results were supported by the demonstration of the effects of the tested treatments on histological changes in kidney tissues. Kidney tissues of DN untreated hamsters (G2) showed cytoplasmic vacuolization of the epithelial lining renal tubules and endothelial lining glomerular tuft as well as protein cast in the lumen of renal tubules. Also, glomerular hypertrophy, mesangial expansion and thickening of glomerular basement membrane were appeared. However, kidney tissues of DN hamsters given metformin (G3), native quercetin (G4) and nano quercetin (G5) showed significant improvement compared with G2. While Kidney sections of DN hamsters given nano quercetin plus metformin (G7) revealed no histopathological alterations.
In general, the results obtained in this study showed the beneficial effects of consuming oral doses of native or nano quercetin with and without metformin. Our results established the remarkable effect of QUNPs most likely by virtue of their small size and characteristics; enhanced bioavailability, absorption, uptake and delivery across membrane. In addition to its small size, the effectiveness was considered to be due to its phytochemical properties and its impressive antioxidant activities. Nonetheless, native quercetin was marginally less effective when used as a singular treatment.