间充质干细胞经PI3K/AKT/mTOR与TGF-β信号调节糖尿病大鼠胰腺和脾脏炎症

Introduction

Diabetes mellitus (DM) is a global health problem characterized by chronic hyperglycaemia. The disease has systemic pro-inflammatory and inflammatory potentials with a consequence of multi-organ complications in the form of neuropathy, nephropathy, and cardiovascular diseases, leading to significant morbidity and mortality¹. Despite advances in medical treatments, the management of diabetes and its complications remain challenging, necessitating the exploration of novel therapeutic approaches^2,3.

PI3K/AKT/mTOR and TGF-β are central signalling nodes in β-cell survival and diabetic pathology. PI3K activation by insulin or growth factors generates PIP3, which recruits and activates AKT; impaired PI3K/AKT signalling in diabetes reduces β-cell survival and insulin secretion⁴.

AKT, phosphorylated at Thr308 and Ser473 upon PI3K activation, promotes β-cell survival by suppressing pro-apoptotic mediators; impaired AKT signalling accelerates β-cell loss⁴.

The mTOR, a downstream AKT effector, governs β-cell mass and insulin secretion; its chronic overactivation contributes to β-cell dysfunction and insulin resistance^5,6. Dysregulation of mTOR signaling, particularly mTORC1, can lead to β-cell dysfunction, insulin resistance, and increased susceptibility to stress, thereby contributing to the pathogenesis of both T1D and T2D^4,7,8,9.

So far, insulin replacement therapy is still the gold standard of management. However, insulin may escalate life-threatening side effects due to failure to replicate the actual physiological modulation of the hormone^10,11,12.

Mesenchymal stem cells (MSC) have emerged as a promising therapeutic option for various diseases, including DM, due to their regenerative capabilities, immunomodulatory effects, and ability to differentiate into multiple cell types¹³. Preclinical studies have demonstrated the potential of MSC to repair damaged tissues, reduce inflammation, and modulate immune responses, making them a viable candidate for treating diabetic complications¹¹.

Current diabetes treatment focuses on slowing disease progression by regulating diabetes-related metabolic and hemodynamic abnormalities¹⁴. Nonetheless, mesenchymal stem cells (MSC) have attracted significant attention as a potential restorative therapy for diabetes and its complications¹⁵. MSC have the ability to develop into many mesenchymal tissue lineages³.

This study aimed to examine the effects of undifferentiated bone marrow-derived MSC on gene expression profiles related to apoptosis, inflammation, and cellular stress in a streptozotocin-induced diabetic rat model, and to evaluate accompanying changes in pancreatic and splenic histology and protein expression.

Methods

Experimental design

The study involved 40 female and 10 male Sprague–Dawley rats. Male served as bone marrow donors. The animals were obtained from Moustafa Rashed Company (Giza, Egypt). Animals were housed in stainless steel cages under standard conditions (light, temperature and free access to food and water). All experimental procedures approved by the Ethics Committee, Faculty of Medicine, Alexandria University (No. 0306187/2023). Animal care and use were in accordance with procedures outlined in the National Institutes of Health Guidelines¹⁶.

Isolation and expansion of mesenchymal stem cells (MSC)

Ten male Sprague–Dawley rats (4–6 months old, weighing 200–250 g) were euthanized, placed in a vertical laminar airflow hood where the humerus and femur were dissected under aseptic conditions, cleaned from connective tissues, and reconstituted in 10 ml isolation medium (ThermoFisher Scientific, Paisley, UK), supplemented with 10% antibiotic–antimycotic (Sigma-Aldrich), performing all steps on ice.

Bone marrow cells were obtained by flushing of long bone with 10 ml of complete culture medium, comprised of αMEM with 20% heat-inactivated fetal bovine serum (FBS, Thermo- Fisher Scientific), 1% antibiotic–antimycotic, and 100 μL-ascorbic acid 2-phosphate (Sigma-Aldrich), and filtered through a 70 μm nylon cell strainer into T-75 tissue culture flasks (Greiner Bio-One International, Kremsmünster, Austria). Cells were incubated at 37 °C and 5% CO₂. Nonadherent cells were removed by changing of medium every 24 h¹⁷.

Characterization of MSC by flow cytometry

Cell morphology was assessed using a phase-contrast inverted microscope (Nikon TSM, Champigny-sur-Marne, France) equipped with a digital camera (DCM 510), and cultured cells were monitored for the characteristic spindle-shaped, fibroblast-like morphology of mesenchymal stem cells.

The immunophenotype of second-passage BM-MSCs was evaluated by flow cytometry using fluorescently labelled monoclonal antibodies against CD90, CD73, CD271, and CD45 (Abcam, Cambridge, UK). Briefly, cells were detached using 0.25% trypsin–EDTA, washed with PBS, and incubated with the monoclonal antibodies for 30 min at room temperature in the dark. The cells were then washed three times with PBS and resuspended in 500 µL of FACS buffer. Flow-cytometric analysis was performed using a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson, USA). A CD45-negative/low population with appropriate side-scatter characteristics was identified as gate G1. Within this gated population, co-expression of CD90/CD73 and CD90/CD271 was subsequently assessed in separate staining panels¹⁸.

Osteogenic differentiation of BM-MSC

In a concentration of 2 × 10⁶ Cells/well, cells of third passage were seeded into a 24-well cell culture plate. At 70–80% confluence, the osteogenic differentiation medium (Stempro) was changed weekly¹⁹.

Animal groups and treatment

This study was conducted on 40 female rats, 4–6 months old, 200–250 gm each. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg)²⁰. The single high dose of streptozotocin was used to generate a reproducible, rapidly developing insulin-deficient model consistent with necrotic β-cell loss and the systemic consequences of hyperglycaemia rather than the gradual, immune-mediated β-cell destruction of the multiple low-dose model, which more closely reflects the inflammatory pathogenesis of human type 1 diabetes^21,22. Fasting blood glucose were recorded at baseline and at the end of the treatment period for all animals. The control group received an intraperitoneal injection of 0.1 M citrate buffer 1 ml/Kg (Sigma Chemical Co., St Louis, MO, USA). Three days later, fasting blood glucose was measured using a blood sample taken from the tip of the tail by Dextrostix/Glucometer (Ames, Elkhart, Indianapolis, IN, USA). Rats having blood glucose of 200 mg/dl were considered hyperglycaemic. Rats were divided into four groups (n = 10) in each:

• Control: non-diabetic.

• Diabetic (DM) received (STZ, 60 mg/kg) in 100 µl/day subcutaneously dimethyl sulfoxide “DMSO” (Sigma Chemical Co., St Louis, MO, USA).

• Insulin-treated diabetic (Insulin), received insulin 5 U/day, 1 U being applied at 13 h and the remaining 4 U at 19 h for 2 weeks²⁰.

• Mesenchymal Stem Cell-treated Diabetic (MSC), each rat received a single IP injected dose of MSC (1 × 10⁶ cells) BM-derived MSC.

At the end of the experiment, up to 12-h fasting blood was obtained from the animal tail vein for measuring fasting blood glucose level using a Dextrostix/Glucometer (Ames, Elkhart, Indianapolis, IN, USA) in all animals. Then, the animals were euthanized and sacrificed by cervical dislocation and specimens from the pancreatic tail and spleen were taken and processed for histopathology and immunohistochemistry studies.

Histopathology and immunohistochemistry

Tissue specimen from spleen and pancreas were fixed in 10% formalin, dehydrated in ascending grades of ethanol, embedded in paraffin, and sectioned at 4 µm thickness. Hematoxylin and eosin (H&E) staining was performed to assess tissue architecture using the optical microscope (Leica DMRBE).

Immunohistochemistry (IHC) was conducted using specific antibodies to evaluate protein expression levels. Paraffin sections were mounted on positively charged glass slides with poly L-lysine. Sections were processed by deparaffinization, rehydration, and blocking of endogenous peroxidase activity, and stained with anti-protein kinase B (anti-Akt, 1:400, Abcam), anti-transforming growth factor beta 3 (anti-TGF-beta-3, 1:400, Abcam), mammalian target of rapamycin (Anti-mTOR, 1:400, Abcam), and anti-phosphoinositide 3-kinases (anti-PI3K, 1:400, Abcam) antibodies according to manufacturer's instructions. Using the Horseradish peroxidase/DAB (ABC) detection immunohistochemistry kit (Abcam, Cambridge, UK). The assayed sections were examined by microscopy. Brown cytoplasmic staining was considered a positive reaction. Control pancreas and spleen sections were used as positive control. For negative control staining, sections were processed in an identical manner but incubated with PBS instead of the primary antibody. No immunoreactivity was present in these sections. The antigen-positive area is brown. Random microscopic fields were captured per sample, and signal intensity was then semi-quantified using The Image Processing and Analysis Java (ImageJ) program²³.

RT-PCR gene expression analysis

Following treatment, pancreatic and splenic tissues were harvested, and RNA was extracted using the TRIzol method (Invitrogen, CA, USA). cDNA synthesis was performed using a reverse transcription kit (ThermoFisher Scientific, Inc), and quantitative PCR was conducted to assess the expression of SRY, C-Jun, P53, BCL2, P38, and Caspase3 genes using gene-specific primers designed with NCBI Primer-BLAST Table 1. GAPDH was used as a housekeeping gene for normalization. On an Applied Biosystems ABI 7500 Fast real-time PCR machine (Applied Biosystems, Foster City, CA, USA), the mRNA was quantified. The SYBR Green fluorescent dye and the Rotor-Gene 6000 Series Software 1.7 were used to assess the melting curve. Using the 2^−ΔΔCt method, the relative RNA expression of the target genes was calculated by comparing the expression level of the target genes to that of the housekeeping gene.

Statistical analysis

Data were analysed using GraphPad Prism for Windows (version 8.0.2). Gene expression data were presented as mean ± standard deviation (SD). Comparisons between groups were made using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

Results

Characterization of bone marrow-derived MSC

Isolated cells adhered to the culture flask and displayed the typical spindle-shaped, fibroblast-like morphology of MSC by the second passage. Flow-cytometric analysis demonstrated a predominantly CD45-negative/low BM-MSC population. Within the G1 gated population, 96.51% of cells co-expressed CD90 and CD73, while 88.85% co-expressed CD90 and CD271, supporting the mesenchymal stromal cell phenotype (Fig. 1a). Osteogenic capacity was verified after three weeks in osteogenic medium, where calcium deposition was demonstrated by positive Alizarin Red staining (Fig. 1b). These findings confirm the mesenchymal identity and multipotency of the isolated cells.

Undifferentiated MSC engraftment to the pancreas and spleen

The sex-determining Region Y (SRY) gene, a marker of male donor-cell engraftment, was significantly expressed in the pancreatic and splenic tissues of the MSC-treated group compared with all other groups (Fig. 2a,b; p < 0.001).

Fasting blood glucose

Three days after streptozotocin injection, fasting blood glucose in the diabetic, insulin- and MSC-treated groups exceeded 200 mg/dl, confirming successful diabetes induction, whereas the control group remained normoglycemic. Across the treatment period, untreated diabetic animals showed sustained hyperglycaemia and reduced body-weight gain compared with controls (fasting blood glucose 450.8 ± 77.12 mg/dl versus 97.32 ± 10.29 mg/dl in controls, p < 0.05). Both insulin and MSC treatment lowered fasting blood glucose relative to the untreated diabetic group (312.8 ± 28.44 mg/dl for insulin and 345.2 ± 51.12 mg/dl for MSC, respectively, p < 0.05).

Cell survival gene expression in pancreatic and splenic tissues

Significant reduction in C-Jun expression in the pancreatic and splenic tissues isolated from the MSC-treated and insulin-treated groups compared to its upregulation in the diabetic rats (Fig. 3a; p < 0.05). P53 mRNA expression was significantly upregulated in pancreatic and splenic tissues of diabetic rats compared to the normal group (Fig. 3b; p < 0.05). MSC treatment resulted in a significant downregulation of P53 expression compared to the diabetic group.

BCL2 mRNA expression in MSC- and insulin treated groups showed a notable upregulation in this anti-apoptotic gene (Fig. 3c; p < 0.05). P38 showed a significant increased expression in the diabetic group compared to normal. MSC and insulin treated groups exhibited downregulation in the p38 gene expression (Fig. 3d; p < 0.001). This indicates their cellular metabolic and inflammatory stress alleviation response. Caspase3 expression was significantly reduced in insulin- and MSC-treated rats compared to diabetic rats (Fig. 3e; p < 0.005).

Histopathological architecture examination of pancreatic and splenic tissues

Examination of H & E staining of the control pancreas showed its lobules composed of closely packed pancreatic acini, the exocrine pancreas and islets of Langerhans. Its lobules are separated by very delicate connective tissue septa dissolved during histological preparation and harbored interlobular ducts and blood vessels. Pancreatic islets appeared as compact pale masses embedded within the lobules among pancreatic acini. Islet cells were arranged as anastomosing cords of polyhedral to rounded shaped cells separated by many blood capillaries. Close observation of pancreatic islets revealed that α cells appeared small with darkly stained nuclei and located at the periphery of the islet while β cells appeared as larger cells with lightly stained nuclei residing the centre of the islets (Fig. 4i-a). In diabetic group, blood vessels were engorged, and pancreatic islets were small, and most islet cells appeared shrunken with densely stained and pyknotic nuclei (Fig. 4i-b). In both MSC- and insulin-treated groups, obvious improvement was noticed so the islet cells appeared almost like the control group, however blood vessels were still congested in insulin-treated groups and there was cellular infiltration (Fig. 4i-c,d).

In H & E sections of the spleen of control group, its parenchyma was composed of splenic pulps with predominant red pulp and minor immune part, the white pulp. White pulp was marked by presence of central arteriole and is branches (Fig. 4ii-a). The parenchyma of the spleen of diabetic group exhibited noticeable distortion of its architecture. White and red pulps showed areas of focal degeneration and the cytoplasm of many cells appeared pale and vacuolated (Fig. 4ii-b). After injection of insulin in insulin treated group, splenic pulps were improved compared with diabetic group except for minimal foci of degeneration, cell vacuolation and some disfigurement in their appearance especially in red pulp (Fig. 4ii-c). Splenic sections of stem cell treated group showed obvious recovery. Both white and red pulp architectures were almost like the control (Fig. 4ii-d).

Immunohistochemistry examination of pancreatic tissue

The protein levels of PI3K and AKT proteins were detected with immunohistochemical staining. Compared with the control group, the protein expression levels of PI3K and AKT in pancreatic sections were reduced significantly in diabetic animals (Fig. 5a,b-i,ii,v; p < 0.01). Pancreatic sections from insulin-treated and MSC-treated diabetic rats showed significant increase in PI3K and AKT protein levels compared to diabetic rats (Fig. 5a,5b-iii,iv,v; p < 0.05). The immunohistochemistry detected levels of mTOR were significantly higher in diabetic and MSC-treated compared to normal control (Fig. 5c-i,ii,iv,v; p < 0.001). Insulin-treated animals showed a significant reduction in mTOR relative to the diabetic and MSC-treated groups (Fig. 5c-iii,iv,v; p < 0.001). In MSC-treated animals, mTOR showed a reduction relative to diabetic animals that did not reach statistical significance. Detection of TGFβ in pancreas sections from diabetic animals and MSC treated diabetic groups showed a significantly high level compared to normal non-diabetic rats (Fig. 5d-i,ii,iv,v; p < 0.001). However, the levels of TGFβ protein were significantly reduced in insulin-treated and MSC-treated diabetic rats compared to the diabetes group (Fig. 5d-iii,iv,v; p < 0.01). These immunohistochemical signals reflect the relative tissue abundance of total (non-phosphorylated) PI3K, AKT, mTOR and TGF-β; they do not report the phosphorylation status of the proteins, and the results are therefore interpreted as MSC-associated changes in protein expression rather than as direct evidence of pathway activation.

Immunohistochemistry examination of splenic tissue

A significant reduction in PI3K and AKT proteins were detected with immunohistochemical staining in splenic tissues isolated from diabetic rats compared to the control group (Fig. 6a,b-i,ii,v; p < 0.01). PI3K and AKT proteins in splenic tissue showed significant higher levels in insulin treated rats compared to diabetic animals, while MSC-treated animals showed a significant increase in PI3K protein and non-significant increase of AKT (Fig. 6a,b-iii,iv,v; p < 0.01).

The expression of mTOR was significantly elevated in the diabetic group compared to the control group (Fig. 6c-i,ii, v; p < 0.001). Treatment with either MSC or insulin resulted in a significant reduction in mTOR expression relative to the diabetic group; however, levels remained significantly higher than those observed in normal animals (Fig. 6c-iii,iv,v; p < 0.001).

In the diabetic group, TGF-β expression was significantly increased compared to the non-diabetic control animals (Fig. 6d-i,v; p < 0.01). Both insulin- and MSC-treated groups exhibited a significant reduction in TGF-β expression (Fig. 6d-iii,iv,v; p < 0.001). However, MSC-treated splenic tissue exhibited significant TGF-β protein levels relative to splenic sections from normal rats.

Discussion

Diabetes mellitus (DM) is a serious metabolic disorder with multisystem chronic inflammation²⁴. Diabetes mellitus affects ~ 11% of adult population worldwide with therapeutic disparities mainly in middle/low-income countries, it is also recognized among the top 10 global causes of death²⁵. Type 1 diabetes is an autoimmune disorder marked by β-cell death and progressive lack of insulin²⁶.

The present study examined the capacity of undifferentiated bone marrow-derived MSC to protect pancreatic and splenic tissues in a rat model of STZ-induced diabetes. MSC treatment was associated with reduced apoptosis, dampened inflammatory signalling, and partial restoration of tissue architecture in both organs, suggesting effects that may extend beyond local pancreatic repair.

Diabetic animals showed the expected metabolic and cellular features of STZ-induced diabetes. Pro-apoptotic markers P53 and Caspase-3 were markedly elevated while BCL2 expression fell sharply. These gene expressions change paralleled clear histological deterioration as islet atrophy, β-cell shrinkage, and inflammatory infiltration which are consistent with previously described patterns of STZ-induced β-cell injury^27,28,29. MSC therapy was associated with an improved tissue apoptotic profile through P53 and Caspase-3 downregulation, BCL2 upregulation, and pancreatic islets architecture preservation. This is consistent with the growing body of evidence that MSC-derived paracrine factors, exosomal cargo, and mitochondrial transfer protect β-cells from oxidative stress^29,30,31,32.

PI3K and AKT protein levels were markedly reduced in diabetic pancreatic and splenic tissues, reflecting disruption of the pro-survival signalling axis that normally sustains β-cell viability and function. MSC treatment was associated with restoration of pancreatic PI3K and AKT and splenic PI3K, whereas splenic AKT was restored mainly by insulin, reinforcing the importance of PI3K/AKT activation for β-cell survival, proliferation, and insulin secretion^4,33,34,35. These findings are consistent with recent reports that activation of PI3K/AKT is a critical mediator of MSC-induced β-cell repair and improved glycaemic control^4,36. Notably, mTOR remained elevated in both diabetic and MSC-treated animals, whereas insulin treatment alone brought levels closer to control values. Whether the persistent mTOR elevation in MSC-treated animals reflects incomplete metabolic recovery or an adaptive response during β-cell repair remains unclear. This is plausible because mTOR has a dual role in β-cells: moderate activity supports growth, protein synthesis, and adaptation to metabolic demand, whereas prolonged activation may contribute to cellular stress and impaired insulin signalling^5,8,37,38. The sustained mTOR expression seen in our study may therefore reflect ongoing recovery rather than persistent injury alone. However, as only total mTOR was measured, further studies assessing phosphorylated mTOR and downstream targets are needed to clarify its functional significance.

We observed marked upregulation of TGF-β, c-Jun, and p38 MAPK in diabetic animals, reflecting the chronic inflammatory and pro-fibrotic milieu associated with sustained hyperglycemia³⁹. Importantly, the inflammatory response in the present study was interpreted from TGF-β protein expression together with the expression of p38 MAPK and c-Jun gene as complementary indicators of inflammation- and stress-related signalling. However, A broader cytokine panel, including IL-1β, IL-6, TNF-α and NF-κB, would give a fuller picture of the inflammatory response and will be considered in as a next step. MSC treatment was associated with reduction of these markers accompanied by tangible histological improvement in both the pancreas and spleen. These findings add to the preclinical evidence that MSC-derived exosomes and paracrine factors can suppress MAPK-driven apoptotic and inflammatory signalling in diabetic target tissues^32,40,41,42.

The splenic findings deserve attention. Diabetic animals showed reduced PI3K/AKT and elevated TGF-β in the spleen, mirroring what was seen in the pancreas and pointing to the systemic immune dysregulation characteristic of DM. MSC treatment partially normalized these parameters, suggesting that the therapeutic effect is not confined to the islets. This is consistent with observations that splenic immune populations contribute to β-cell homeostasis, and that splenic injury or removal can predispose to glucose intolerance^43,44. These results raise the possibility that MSC restore immune tolerance partly through their effects on splenic microarchitecture and paracrine cytokine networks which represent a mechanism that has received less attention than pancreatic repair.

The current study collectively supports a mechanistic framework for MSC action in STZ-induced diabetes. Three interconnected pathological axes are simultaneously engaged: (i) PI3K/AKT signalling, which is suppressed in diabetic β-cells and associated with partial restor by MSC, is a recognized driver of β-cell survival, proliferation, and insulin secretion; (ii) the TGF-β/p38 MAPK/c-Jun inflammatory cascade, which drives fibrosis and apoptosis in diabetic target organs, is attenuated by MSC through paracrine and exosomal mechanisms; and (iii) the BCL2/Caspase-3/P53 apoptotic balance, shifted markedly towards cell death in diabetic animals, is restored towards survival following MSC treatment. The observation that these changes occur in both the pancreas, and the spleen identifies systemic immunomodulation as a distinct component of the therapeutic response, beyond the local islet-protective effects that have been the primary focus of prior work. The persistent mTOR elevation in MSC-treated animals is a specific finding that warrants follow-up, as it may reflect a compensatory anabolic state in recovering β-cells rather than ongoing pathology. Because these pathway readouts are based on protein abundance and gene expression rather than phosphorylation or loss-of-function analyses, our findings interpreted as correlated changes consistent with MSC-mediated modulation, rather than evidence of direct pathway regulation. Overall, the findings support further preclinical evaluation of MSCs while recognizing that the observed molecular changes do not establish direct causal regulation of these pathways.

Several limitations should be considered, along with directions for future work. We used a single MSC dose and follow-up time and did not assess long-term cell engraftment or persistence. Protein expression was evaluated semi-quantitatively by IHC, without measuring phosphorylated AKT or mTOR, so pathway activation could not be confirmed directly. Future studies should therefore explore different MSC doses, longer follow-up, functional pathway markers, β-cell mass, and a broader inflammatory profile, ideally using a multiple low-dose STZ diabetes model that better reflects the inflammatory component of diabetes.

Data availability

Data will be available upon reasonable request.

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Funding

The authors did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors for this research.

Author information

Authors and Affiliations

1. Department of Anatomy, Faculty of Medicine, University of Tabuk, Tabuk, Saudi Arabia

   Salwa Fares Ahmed & Amira H. Eltrawy

2. Department of Histology, Faculty of Medicine, Assiut University, Assiut, Egypt

   Salwa Fares Ahmed & Asmaa F. A. Dawood

3. Department of Pharmacology, Faculty of Medicine, Tanta University, El Gharbia, Tanta, Egypt

   Sabah H. El-Ghaiesh

4. Department of Anatomy and Embryology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

   Amira H. Eltrawy

5. Department of Biological Sciences, College of Science, University of Jeddah, Jeddah, Saudi Arabia

   Fatima A. Jaber

6. Department of Histopathology, Prince Sultan Medical Military City, Riyadh, Saudi Arabia

   Ranim F. Alderbas

7. Department of Medical Physiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

   Nermin I. Rizk

8. Department of Pediatric, Faculty of Medicine, Menoufia University, Menoufia, Egypt

   Nagwa G. M. Gad

9. Department of Biomedical Science, Faculty of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia

   Asmaa F. A. Dawood

Authors

1. Salwa Fares Ahmed
2. Sabah H. El-Ghaiesh
3. Amira H. Eltrawy
4. Fatima A. Jaber
5. Ranim F. Alderbas
6. Nermin I. Rizk
7. Nagwa G. M. Gad
8. Asmaa F. A. Dawood

Contributions

S.F.A., S.H.E., A.F.A.D., A.H.E., F.A.J., R.F.A.; N.I.R. conceptualization, study design, methodology, S.F.A., A.F.A.D., A.H.E., F.A.J., R.F.A.; Histopathology processing, immunohistochemistry staining, and image analysis, N.I.R. & N.G.M.G.; PCR, Biochemical analyses, RT-PCR work, S.H.E. data analysis, visualization, S.H.E., S.F.A., A.H.E., N.I.R., A.F.A.D., N.G.M.G.; Manuscript drafting, figure preparation, and formatting of results and references, All authors contributed to writing, critically revising the manuscript for intellectual content, and approved the final version of the manuscript for submission.

Corresponding author

Correspondence to Sabah H. El-Ghaiesh.

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Competing interests

The authors declare no competing interests.

ARRIVE guidelines compliance

This study was designed, conducted, and reported in accordance with the ARRIVE 2.0 guidelines for reporting animal research. A completed ARRIVE checklist is provided as supplementary material.

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Ahmed, S.F., El-Ghaiesh, S.H., Eltrawy, A.H. et al. Mesenchymal stem cells modulate pancreatic and splenic inflammation through PI3K/AKT/mTOR and TGF-β signalling in diabetic rats. Sci Rep 16, 32659 (2026). https://doi.org/10.1038/s41598-026-72653-1

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• Received: 06 June 2026

• Accepted: 17 September 2026

• Published: 16 October 2026

• Version of record: 16 October 2026

• DOI: https://doi.org/10.1038/s41598-026-72653-1

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