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The present study was undertaken to investigate the possible therapeutic mechanisms of BM-MSCs infusion against experimentally induced type 2 diabetic (T2D) rat model.
To achieve this aim, twenty young (6-8 weeks old) male Wistar rats weighing 60-75g were used to derive BM-MSCs cultures. BM-MSCs were isolated, cultured, counted and trypsinized, and then they were identified by flow cytometry via detecting positive BM-MSCs markers (CD44) as well as hematopoietic markers (CD19).
In the present study the isolated BM-MSCs showed the fibroblastic morphology that is characteristic of MSCs. In addition, analysis by flow cytometry demonstrated that BM-MSCs were 91.8% pure for CD44. The percentage of contaminated populations of hematopoietic stem cells positive for CD19 was 8.9%.
Thereafter, thirty rats were divided into 3 groups (10 rats each) as follows: group Ӏ (Normal Control): normal rats were i.p. injected with sterile saline; group ӀӀ (T2D): normal rats were fed with HFD followed by a single i.p. injection with STZ; group ӀӀӀ (T2D + BM-MSCs): diabetic rats were treated by a single i.v. injection of 2 × 106 BM-MSCs prepared in sterile saline. Rats were observed for a further 5 weeks (the experimental period) and the fasting blood glucose levels from the tail vein were monitored regularly using a digital glucometer.
At the end of the experimental period, the animals were fasted for 8 hours, weighted, and the blood samples were taken from the retro-orbital venous plexus under light ether anesthesia with potassium oxalate/sodium fluoride mixture for separation of plasma or without this mixture for separation of serum. Pancreas, liver, and skeletal muscles were collected.
For histological examination pancreatic specimens from the different groups were fixed in 10% formalin solution and stained by hematoxylin and eosin to be examined under the electric light microscope.
Fasting plasma glucose and fasting serum insulin were assayed, the indices of HOMA-IR and HOMA-β were calculated, complete lipid profile was assessed with calculating atherogenic indices 1 & 2. In addition, serum leptin and adiponectin were measured and A/L ratio was calculated. Furthermore, the expressions of the glucokinase in liver, glycogen synthase in liver and skeletal muscles, and IGF-1R in pancreas were quantified using qPCR. Finally the contents of GK and GS enzymes were assayed in liver and skeletal muscles homogenates.
Induction of T2D in rats by HFD followed by single i.p administration of STZ elevated significantly fasting plasma glucose and fasting serum insulin levels (430.14 and 127.74%, respectively), compared to the normal control rats. On the other hand, treatment of with BM-MSCs reduced significantly fasting blood glucose and serum insulin levels, compared type 2 diabetic-rats, while still significantly higher than the normal levels (46.25 and 31.75%, respectively).
Calculating HOMA-IR & HOMA-β indices of type 2 diabetic-rats showed a significant increase in HOMA-IR index (1108.89%) with a significant reduction in HOMA-β index (57.2%). However, BM-MSCs therapy maintained their values to the normal ratios.
Furthermore type 2 diabetic-rats had significant elevations in the serum levels of TC (103.49%), TAG (64.12%), VLDL-C (64.15%), and LDL-C (284.82%), as well as atherogenic indices 1 & 2 (202.16 and 476.19%, respectively), in association with a significant reduction in serum HDL-C level (31.3%), compared to the normal control rats. On the other hand, treatment of type 2 diabetic-rats with BM-MSCs normalized TC, TAG, VLDL-C, LDL-C, atherogenic indices 1 & 2. In addition, BM-MSCs therapy improved serum HDL-C level, compared to type 2 diabetic-rats, but was still significantly lower than the normal level (13.41%).
There were a slight significant reduction in the serum adiponectin level in HFD/STZ and T2DM+BM-MSCs groups (16.2 and 13.64%, respectively), compared to the normal control rats. Furthermore, serum leptin level was increased significantly in type 2 diabetic-rats (97.74%), compared to the normal control rats. Meanwhile, treatment of type 2 diabetic-rats with BM-MSCs normalized serum leptin level. Adiponectin/ leptin (A/L) ratio showed a marked significant reduction (57.32%) in HFD/STZ-induced type 2 diabetes; and this reduction was significantly improved after BM-MSCs therapy (23.98%), compared to the normal control group.
There were significant down regulations in the relative expression of GK and GS in liver of HFD/STZ-induced T2D, GS in the skeletal muscles and IGF-1R in pancreas of HFD/STZ-diabetic rats compared to the normal control rats. On the other hand, these genes were significantly up-regulated after BM-MSCs therapy compared to both HFD/STZ and normal control groups.
There were significant reductions in the contents of liver GK (28.75%), liver GS (25.15%), and skeletal muscle GS (43.46%) in HFD/STZ group, compared to the normal control rats. Treatment of type 2 diabetic-rats with BM-MSCs increased significantly the contents of liver GK (23.32%), liver GS (25.95%), compared to both HFD/STZ and normal control groups, while BM-MSCs normalized muscle GS content.
The histological examination of the H&E-stained pancreas sections of the normal control group (Gr.1) under the light microscope revealed normal histological structure of the islands of Langerhans as endocrine portion as well as the acini with the duct system as exocrine. On the other hand, pancreas sections from HFD/STZ-induced T2D (Gr.2) reported the presence of atrophy in a diffuse manner all over the islands of Langerhans cells, associated with severe congestion in the stromal interlobular blood vessels. The interstitial stroma showed fat vacuoles as well as inflammatory cells infiltration. Pancreas sections from T2D rats treated with BM-MSCs (Gr.3) showed that the islands of Langerhans cells were nearly intact but smaller in size than the normal control one with the presence of cystic dilatation in the duct system.