Int J Stem Cells.  2020 Mar;13(1):163-175. 10.15283/ijsc18066.

Effect of Stem Cells, Ascorbic Acid and SERCA1a Gene Transfected Stem Cells in Experimentally Induced Type I Diabetic Myopathy

• Zickri MB
• Sadek EM
• Fares AE
• Heteba NG
• Reda AM

• KMID: 2500575
• DOI: http://doi.org/10.15283/ijsc18066

Abstract

Background and Objectives
Sarco/endoplasmic reticulum Ca2＋-ATPase (SERCA) inhibition was proved in streptozotocin (STZ)-diabetic rats. The present study aimed at investigating and comparing the therapeutic effect of bone marrow mesenchymal stem cells (BMMSCs), BMMSCs combined with ascorbic acid (AA) and SERCA1a gene transfected BMMSCs in induced type I diabetic myopathy of male albino rat.
Methods and Results
54 rats were divided into donor group of 6 rats for isolation, propagation and characterization of BMMSCs and SERCA1a transfected BMMSCs, groups I∼V 48 rats. Group I of 8 control rats, group II (Diabetic) of 10 rats given STZ 50 mg/kg intraperitoneal, group III (BMMSCs) of 10 rats given STZ and BMMSCs intravenous (IV), group IV (BMMSCs and AA) of 10 rats given STZ, BMMSCs IV and AA 500 mg/kg and group V (SERCA 1a transfected BMMSCs) of 10 rats given STZ and SERCA1a transfected BMMSCs IV. The rats were sacrificed after 8 weeks. Gastrocnemius specimens were subjected to biochemical, histological, morphometric and statistical studies. Diabetic rats revealed inflammatory and degenerative muscle changes, a significant increase in blood glucose level, mean DNA fragmentation and mean MDA values and a significant decrease in mean GSH and catalase values, area of pale nuclei, area% of CD105 and CD34 ＋ve cells, SERCA1a protein and gene values. The morphological changes regressed by therapy. In group III significant decrease in DNA fragmentation and MDA, significant increase in GSH and catalase, significant increase in the mean area of pale nuclei, area % of CD105 and CD34 ＋ve cells versus diabetic group. In group IV, same findings as group III versus diabetic and BMMSCs groups. In group V, same findings as group IV versus diabetic and treated groups. Western blot and PCR proved a mean value of SERCA1a protein and gene comparable to the control group. Mean calcium concentration values revealed a significant increase in the diabetic group, in BMMSCs and AA group versus control and SERCA1a group.
Conclusions
SERCA1a transfected BMMSCs proved a definite therapeutic effect, more remarkable than BMMSCs combined with AA. This effect was evidenced histologically and confirmed by significant changes in the biochemical tests indicating oxidative stress, muscle calcium concentration, morphometric parameters and PCR values of SERCA1a.

Keyword

Diabetes; Ascorbic acid; Skeletal muscle; BMMSCs; SERCA1a; Myopathy

Figure

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  Fig. 1 Showing: (a) CD105 ＋ve spindle (s) cells. (b) CD34 −ve immunoreactivity (arrows), (Phase contrast microscopy ×100). (c) Immunophenotyping of BMMSCs 98% are ＋ve for CD105 (Flow Cytometry). (d) Transfected cells appearing mostly as spindle fluroscent labeled cells (GFP ×100). (e) Immunophenotyping of transfected BMMSCs 94% are GFP labeled (Flow Cytometry).

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  Fig. 2 H&E stain. Group I showing (a) fibers (mf) exhibiting peripheral nuclei and acidophilic sarcoplasm, (×200). (b) Fibers exhibiting regular transverse striations (St), pale nuclei (N) and satellite cells (SC), (×400). Group II showing (c) multiple atypical widely separated fibers (arrowheads), few disrupted fibers (wavy arrow), some dark (d) and few central nuclei (n), (×200). (d) Multiple disrupted widely separated atypical fibers (wavy arrows), few central pale nuclei (n) and multiple macrophages (m) in between (×400).

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  Fig. 3 H&E stain. Group III showing (a) organized fibers with flat nuclei (stars), some separated fibers (arrowheads) and a congested vessel (c) (×200). (b) Fibers with few pale peripheral nuclei (N), few pale central nuclei (n) and striations (St) in some parts of the sarcoplasm (2400). Group IV showing (c) some fibers with rows of central oval nuclei (n), multiple flat nuclei (stars) and some mononuclear cells (arrowheads) (2200). (d) Some fibers with rows of central pale nuclei (n), others with minimal separation of myofibrils (wavy arrows), striations (St) in multiple areas of the sarcoplasm and flat dark nuclei (stars) (2400). Group V showing (e) some regular fibers with rows of central nuclei (n), other fibers with peripheral nuclei (N) and two congested vessels (c) (2200). (f) Rows of pale central nuclei (n), obvious striations (St), a central flat dark nucleus (star) and focal separation of myofibrils (arrowheads) (2400).

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  Fig. 4 PKH26 (×100) showing in (a) group III multiple fluorescent labeled cells (arrows) among the fibers. (b) Group IV some labeled cells (arrows) among the fibers. Pb (×400) (c) group V few ＋ve spindle (s) cells among the fibers. GFP (×100) (d) few fluorescent labeled cells.

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  Fig. 5 CD105 immunostaining, (×400) showing in (a) group I −ve immunoexpression among the fibers (mf) and in a vessel (v). (b) Group II some ＋ve spindle cells in the CT near a vessel (v) (arrows) and near obviously vacuolated fibers (mf) (arrowheads). (c) Group III multiple ＋ve cells among the fibers (mf) (arrowheads) and few in the CT (arrows). (d) Group IV multiple ＋ve cells in the CT between the fibers (mf) (arrows) and few on a fiber (arrowhead). (e) Group V some ＋ve cells in two vessels (v) (arrows) and few on a fiber (mf) (arrowhead).

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  Fig. 6 CD34 immunostaining, (×400) showing in (a) group II few ＋ve flat cells in the CT between some fibers (mf) (arrows) and in the lining of a vessel (v). (b) Group III some ＋ve flat cells at the periphery (arrows) and at the center (arrowhead) of some fibers. (c) Group IV multiple ＋ve flat cells at the periphery (arrows), at the center (arrowheads) of some fibers, in the CT (stars) and in the lining of a vessel (v). (d) Group V numerous ＋ve flat cells (arrows) at the periphery of fibers.

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  Fig. 7 (a) Western blot showing SERCA1a protein detected and analyzed (normalized to actin) in gastrocnemius tissue from groups I to V (b) mean density of SERCA1a (c) mean PCR values of SERCA1a gene, (a) and (c) denoted significant increase in groups I and V versus other groups and in groups III and IV versus group II.

Reference

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