# Therapeutic Effect of Human Umbilical Cord Mesenchymal Stromal Cells Loaded With miR‐9‐5p on Hypoxic‐Ischemic Brain Damage in Neonatal Rats

## TL;DR

This study shows that human umbilical cord stem cells loaded with miR-9-5p can reduce brain damage and improve recovery in newborn rats with hypoxic-ischemic brain injury.

## Contribution

The study demonstrates that miR-9-5p enhances the therapeutic potential of hUC-MSCs for neonatal brain injury through immune modulation and neuroprotection.

## Key findings

- miR-9-5p-hUC-MSCs significantly reduced cerebral infarct volume compared to the HIBD model group and Ad-hUC-MSCs.
- miR-9-5p-hUC-MSCs improved both short-term and long-term neurobehavioral outcomes in HIBD rats.
- miR-9-5p-hUC-MSCs more effectively downregulated apoptotic proteins Beclin-2 and Caspase-3 in hippocampal tissue.

## Abstract

To investigate the therapeutic mechanisms of miR‐9‐5p‐overexpressing human umbilical cord mesenchymal stromal cells (hUC‐MSCs) in neonatal rat models of hypoxic‐ischemic brain damage (HIBD).

Fresh neonatal umbilical cords were collected to isolate and culture human umbilical cord mesenchymal stromal cells (hUC‐MSCs). Recombinant adenovirus was used to amplify miR‐9‐5p and transduce hUC‐MSCs, generating miR‐9‐5p‐overexpressing cells. Functional assessments included: ELISA to evaluate secretory function (e.g., neurotrophic and anti‐inflammatory factors), real‐time cell analysis to measure proliferation capacity, Transwell and Dunn chamber assays to assess chemotactic migration ability. Healthy 7‐day‐old Sprague‐Dawley (SD) rats of both sexes were randomly allocated into four groups (n = 12/group, with 4 rats per group assigned to TTC staining, Western blot, or Morris water maze assay, respectively): Sham‐operated control group (mock surgery), Hypoxic‐ischemic brain damage (HIBD) model group, miR‐9‐5p‐hUC‐MSCs treatment group, and Adenovirus‐transduced hUC‐MSCs (Ad‐hUC‐MSCs) treatment group. The HIBD model was induced in groups 2–4. At 24 h post‐modeling, 1×106 miR‐9‐5p‐hUC‐MSCs or Ad‐hUC‐MSCs were transplanted into the left lateral ventricle (injured side) of the pups. Neurological function was evaluated 48 h later using righting reflex tests for short‐term neurobehavioral assessment. Subsequently, 4 rats per group were sacrificed for 2,3,5‐triphenyltetrazolium chloride (TTC) staining to quantify cerebral infarction volume. Hippocampal tissues from another 4 rats per group were analyzed by Western blot for Beclin‐2 and Caspase‐3 protein expression. The remaining 4 rats per group underwent 28‐day Morris water maze testing for long‐term neurobehavioral evaluation.

Spindle‐shaped and polygonal adherent cells emerged within 3–5 days following umbilical cord tissue block inoculation, with flow cytometric analysis confirming their identity as mesenchymal stromal cells (MSCs). Compared to the Ad‐hUC‐MSCs treatment group, miR‐9‐5p enhanced the secretion of neuroreparative and anti‐inflammatory factors (e.g., NGF, BDNF, IL‐6) in hUC‐MSCs while suppressing pro‐inflammatory cytokines (e.g., IL‐1, IL‐2) (p < 0.05). Furthermore, miR‐9‐5p significantly promoted hUC‐MSCs proliferation and augmented the chemotactic migratory capacity of miR‐9‐5p‐hUC‐MSCs. At 48 h post‐transplantation in the miR‐9‐5p‐hUC‐MSCs group, the sham‐operated controls showed no detectable cerebral infarction, whereas the model group exhibited distinct pale infarct foci occupying 33.15% ± 4.38% of total brain volume (vs. controls, p < 0.05), indicating severe cerebral injury. Both miR‐9‐5p‐hUC‐MSCs and Ad‐hUC‐MSCs treatments markedly reduced infarct volumes to 14.85% ± 2.79% and 19.11% ± 4.57%, respectively, with the miR‐9‐5p‐hUC‐MSCs group demonstrating a statistically superior therapeutic effect compared to Ad‐hUC‐MSCs (p < 0.05). Transplantation of either Ad‐hUC‐MSCs or miR‐9‐5p‐hUC‐MSCs significantly improved short‐ and long‐term neurobehavioral outcomes in hypoxic‐ischemic brain damage (HIBD) rats. At 48 h post‐HIBD induction, upregulated expression of Beclin‐2 and Caspase‐3 proteins was observed in brain tissue. Notably, these elevated protein levels were attenuated following treatment with miR‐9‐5p‐hUC‐MSCs or Ad‐hUC‐MSCs.

MiR‐9‐5p enhances the secretion of immunomodulatory factors and improves the migratory and proliferative capacities of hUC‐MSCs. Overexpression of miR‐9‐5p promotes in vivo homing of hUC‐MSCs, which mitigate cerebral injury and exert neuroprotective and reparative effects through dual mechanisms: modulating immune responses and providing neurotrophic support. Furthermore, hUC‐MSCs significantly reduce cerebral infarct volume in hypoxic‐ischemic brain damage (HIBD) rats and downregulate levels of apoptotic proteins (Beclin‐2 and Caspase‐3) in brain tissue, demonstrating potent cerebroprotective effects.

To address the lack of effective therapies for neonatal hypoxic‐ischemic brain damage (HIBD), this study explored the therapeutic potential of miR‐9‐5p‐overexpressing human umbilical cord mesenchymal stromal cells (hUC‐MSCs). hUC‐MSCs were isolated, cultured, and transduced with miR‐9‐5p via recombinant adenovirus. Functional assays confirmed that miR‐9‐5p enhanced hUC‐MSCs' secretion of neurotrophic/anti‐inflammatory factors (NGF, BDNF, IL‐6) while suppressing pro‐inflammatory cytokines (IL‐1, IL‐2), and promoted hUC‐MSCs'. proliferation and chemotactic migration. Seventy‐two 7‐day‐old SD rats were randomized into four groups (sham, HIBD model, Ad‐hUC‐MSCs, miR‐9‐5p‐hUC‐MSCs). HIBD models were induced by left carotid artery ligation plus hypoxia, followed by intracerebroventricular transplantation of 1 × 106 modified hUC‐MSCs at 24 h post‐modeling. Key results: ① miR‐9‐5p‐hUC‐MSCs significantly reduced cerebral infarct volume (14.85 ± 2.79% vs. 33.15 ± 4.38% in model group, p < 0.05) and outperformed Ad‐hUC‐MSCs (19.11 ± 4.57%, P < 0.05); ② miR‐9‐5p‐hUC‐MSCs improved short‐term (righting reflex) and long‐term (Morris water maze) neurobehavioral outcomes; ③ miR‐9‐5p‐hUC‐MSCs more effectively downregulated hippocampal apoptotic proteins (Beclin‐2, Caspase‐3) vs. Ad‐hUC‐MSCs. These findings demonstrate that miR‐9‐5p enhances hUC‐MSCs' neuroprotective efficacy via modulating immune responses, promoting neurotrophy, and inhibiting apoptosis, providing a promising therapeutic strategy for neonatal HIBD.