Peripheral blood samples were collected from 42 children diagnosed with HSP at the Department of Pediatrics, Affiliated Hospital of Nantong University, between June 2023 and December 2024. Samples from 42 age-matched healthy children served as controls. Diagnosis of HSP was based on the 2010 EULAR/PRINTO/PRES criteria [12]. Healthy controls had no recent infections (within 4 weeks), immunodeficiency, genetic disorders, or major systemic diseases. Written informed consent was obtained from legal guardians. The study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University (Approval No.: 2022-K025-01). All patients received standard symptomatic and supportive treatments; severe cases with organ involvement received corticosteroids. Long-term progression to renal failure was not evaluated in this cross-sectional cohort.

Human umbilical vein endothelial cells (HUVECs) and human umbilical cord mesenchymal cells (hUC-MSCs) were obtained from the Department of Obstetrics and Gynecology, Affiliated Hospital of Nantong University. hUC-MSCs had been authenticated for characteristic phenotypic and differentiation potential [13].

HUVECs were cultured in DMEM/F12 medium with 10% heat-inactivated serum from healthy children (Ctrl) or from HSP patients (HSP). After 24 h of stimulation, HSP-conditioned HUVECs were co-cultured with hUC-MSCs using a Transwell system equipped with a 0.4-μm pore size polyester membrane insert (BD Biosciences, California, USA), allowing paracrine interaction without direct cell–cell contact (HSP+MSC). To investigate the involvement of STAT3 signaling, HUVECs were pretreated with colivelin TFA (MCE, New Jersey, USA) at an optimized concentration for 1 h prior to co-culture (HSP+MSC+TFA).

For immune cell experiments, human CD4⁺ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using magnetic-activated cell sorting (MACS) with a CD4⁺ T cell isolation kit (Miltenyi Biotec, California, USA), according to the manufacturer’s protocol. Purified CD4⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated human serum. Cells were then exposed to Ctrl or HSP serum for 24 h, followed by co-culture with hUC-MSCs using the same Transwell system described above. Where indicated, CD4⁺ T cells were treated with colivelin TFA for 1 h prior to co-culture.

To assess STAT3 signaling responses in hUC-MSCs, hUC-MSCs were cultured alone and incubated with Ctrl serum, HSP serum, or HSP serum in the presence of colivelin TFA for 24 h. Cells were then harvested for protein extraction and subsequent analysis of IL-6, STAT3, and p-STAT3 expression.

Forty 4-week-old male Sprague-Dawley rats were obtained from the Standardized Experimental Animal Breeding Base, Nantong University. An HSP animal model was induced using ovalbumin (OVA, Solarbio, Beijing, China) emulsion and saline injections [14]. Rats were randomly assigned to four groups (n = 10/group): Healthy control (Ctrl), HSP model (HSP), hUC-MSCs treatment (HSP+MSC), and hUC-MSCs + colivelinTFA treatment (HSP+MSC+TFA). Groups HSP, HSP+MSC, and HSP+MSC+TFA received intraperitoneal OVA emulsion (10 mg/kg) weekly for 3 weeks, followed by OVA challenge via tail vein and intradermal injections. The HSP+MSC group additionally received hUC-MSCs (2 × 106 cells/mL, 1 mL, via tail vein) every 3 days after hypersensitivity induction. The HSP+MSC+TFA group also received colivelin TFA (0.1 mg/mL, 10 mL/kg, intraperitoneally) after each hUC-MSCs injection (Supplementary 1A). The dosing regimen was selected based on preliminary experiments and supported by previously published studies [15]. At the end of modeling, rats were euthanized, and kidney, joint, and intestinal tissues were collected. All procedures were approved by the Animal Welfare Ethics Committee (Approval No.: S20230321-002).

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Lymphoprep, Thermo Fisher, USA). Total RNA was extracted using Trizol reagent, and reverse transcription was performed with the PrimeScript™ RT Reagent Kit (TaKaRa, Cat #RR037A, Kusatsu, Japan). Quantitative PCR was conducted using SYBR™ Green Master Mix (Applied Biosystems, Shanghai, China) with 40 amplification cycles. GAPDH served as the internal control. Primer sequences are listed in Table 1.

Serum concentrations of IL-17 and IL-10 were quantified using commercial human ELISA kits (IL-17A and IL-10, Thermo Fisher Scientific, California, USA). Serum samples were diluted 1:4 in the provided assay diluent. A standard curve was generated using the recombinant cytokine standard (range: 0–400 pg/mL). Briefly, 100 µL of diluted samples or standards were added to pre-coated 96-well plates and incubated for 2 h at room temperature on a plate shaker (300 rpm). After four washes, 90 µL of detection antibody was added per well and incubated for 1 h. Following another wash step, streptavidin-HRP was added, incubated for 30 min in the dark, and the plate was washed again. TMB substrate was added, incubated for 15 min in the dark, and the reaction was stopped with 50 µL of stop solution per well. Absorbance was measured at 450 nm with a 570 nm reference using a microplate reader. Cytokine concentrations were interpolated from the standard curve via a 4-parameter logistic (4PL) regression model.

Tissue samples (kidney, joint, and intestine) were fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4 °C for 24 h, washed, and processed through graded ethanol, xylene, and paraffin wax under standard protocols. Sections (4 μm) were cut using a rotary microtome (Leica RM2235, Wetzlar, Germany), mounted on coated slides, dried at 37 °C overnight, and baked at 55 °C for 2 h before staining. For H&E staining, sections were deparaffinized in xylene (two 10-min incubations; Sinopharm AR grade, Sigma-Aldrich ACS grade) and rehydrated through a graded ethanol series (100%, 90%, 80%, 70%; 5 min each, Fisher Scientific HPLC grade). After rinsing in deionized water, nuclei were stained with Harris hematoxylin solution from a commercial H&E staining kit (Beyotime Biotechnology, Shanghai, China) for 5 min, rinsed, and cytoplasm was counterstained with the accompanying eosin Y solution (1 s). Staining quality was verified microscopically after each step.

Sections were dehydrated through an ascending ethanol series (70% to 100%; 5 min each), cleared in xylene (two 15-min changes), and mounted with neutral balsam. Slides were examined under a light microscope, and representative images were captured digitally for analysis.

Total protein was extracted with RIPA buffer, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked and incubated with primary antibodies against IL-6 (Proteintech, Wuhan, China, 1:1000), RORγt (Affinity Biosciences, Cincinnati, USA, 1:1000), STAT3 (Proteintech, Wuhan, China, 1:1000), p-STAT3 (Proteintech, Wuhan, China, 1:1000), and GAPDH (Proteintech, Wuhan, China, 1:10000), followed by HRP-conjugated secondary antibodies (Sangong Biotech, Shanghai, China, 1:10000). Bands were visualized and quantified. Each experiment was independently repeated at least three times.

PBMCs and rat splenocytes were isolated, red blood cells lysed, and lymphocytes enriched. Cells were surface-stained with FITC-conjugated anti-human CD4 antibody (Biolegend, California, USA) or PerCP/Cy5.5-conjugated anti-rat CD4 antibody (Biolegend, California, USA) combined with Fc block, and stimulated for 4 h at 37°C (with Leukocyte Activation Cocktail, BD, New Jersey, USA). For intracellular staining, cells were fixed, permeabilized, and stained with APC-conjugated anti-FOXP3 (Biolegend, California, USA) and PE-conjugated anti-IL-17A (Biolegend, California, USA) for 30 min in the dark. After washing, cells were resuspended in PBS, filtered, and analyzed on a BD FACS Aria III flow cytometer. Data were acquired on a BD FACS Aria III cytometer and analyzed with FlowJo v10.8 software.

Colivelin TFA was serially diluted in basal culture medium to concentrations of 0, 2.5, 5, 7.5, 10, and 12.5 mM. HUVECs were harvested using 0.25% trypsin-EDTA, resuspended in complete medium, and seeded into a 96-well plate at a density of 5 × 10³ cells/well (100 µL/well). After overnight incubation (37°C, 5% CO2), the medium was replaced with 100 µL of fresh medium containing the corresponding colivelin TFA concentration. Following a 24-h treatment under standard conditions, 10 µL of CCK-8 reagent (Dojindo, Kamimashiki-gun, Japan) was added to each well. The plate was protected from light and incubated at 37 °C for 3–4 h. Absorbance at 450 nm was measured using a microplate reader. Each concentration was tested in quadruplicate or quintuplicate, and blank control wells (medium only) were included for background subtraction. The mean optical density (OD) of replicates was used for analysis.

HUVECs were seeded in 6-well plates at 5 × 105 cells/well and cultured until 90% confluent. A standardized cross-shaped wound was created using a sterile 200 µL pipette tip (orifice diameter: 0.4 mm) by dragging the tip vertically and horizontally across the monolayer to generate two perpendicular scratches spaced 1 mm apart. Wounded monolayers were washed three times with pre-warmed PBS, then incubated in 2 mL of serum-free DMEM/F12 medium supplemented with 1% penicillin–streptomycin. Phase-contrast images of the initial wound (0 h) were captured at 200 × magnification using an inverted light microscope. After 24 ± 0.5 h incubation under standard conditions (37 °C, 5% CO₂), cells were fixed with 4% paraformaldehyde and stained with 0.1% (w/v) crystal violet. The wound area at 24 h was imaged under identical settings and quantified using ImageJ software.

Data are presented as mean ± SEM or mean ± SD from at least three independent experiments. Normality of continuous variables was assessed using the Shapiro-Wilk test (all P > 0.05), Two-group comparisons were performed with Student's t-test, and multi-group comparisons with one-way ANOVA followed by post hoc testing. A p-value < 0.05 was considered statistically significant.

The authors analyzed PBMCs from 42 pediatric HSP patients and 42 healthy controls (demographic characteristics in Table 2; no baseline differences between groups). Flow cytometry revealed a significant increase in Th17 cells and a reduction in Tregs among CD4+ T cells from HSP patients (Figure 1A-E). Consequently, the Th17/Treg ratio was markedly elevated in the HSP group (Figure 1F). Consistent with these findings, RT-qPCR showed increased expression of the Th17 transcription factor RORC and decreased expression of the Treg transcription factor FOXP3 in patient PBMCs (Figure 1G-H). Cytokine profiling further confirmed this imbalance: serum IL-17 was elevated, while IL-10 was reduced in HSP patients compared with controls (Figure 1I-J).

Figure 1.

Th17/Treg imbalance in HSP children. Flow cytometry and molecular analysis of Th17 and Treg subsets in PBMCs from healthy controls (Ctrl, n = 42) and HSP patients (HSP, n = 42). (A–B) Representative flow cytometry gating strategy from one subject. (A) Singlets gate based on FSC-A/FSCH scatter plot. (B) CD4⁺ T cells gate (FITC-A⁺, 99.1% of singlets). (C-D) Identification of Th17 (CD4⁺IL-17A⁺, upper right quadrant) and Treg (CD4⁺FOXP3⁺, lower right quadrant) subsets. (E) Percentage of Th17 and Treg cells among CD4+ T cells (n = 42/group). (F) Th17/Treg ratio. (G–H) Relative mRNA expression of RORC and FOXP3 (n = 42/group). (I–J) Serum concentrations of IL-10 and IL-17 (n = 42/group). Data are means ± SD, * P < 0.05; *** P < 0.001.

HUVECs cultured with HSP serum displayed abnormal morphology and impaired wound closure, consistent with severe endothelial dysfunction (Figure 2A–B). Co-culture with hUC-MSCs restored normal cell morphology and significantly enhanced migration (Figure 2A–C). However, STAT3 reactivation with colivelinTFA abolished these benefits, reinstating both morphological abnormalities and defective wound healing.

Figure 2.

hUC-MSCs restore endothelial morphology and migration by suppressing STAT3. (A) Representative images of HUVECs cultured under the indicated conditions. Scale bar = 100 µm. (B) Wound healing assay images at 0 h and 24 h. Scale bar = 100 µm. (C) Quantification of wound closure. Ctrl: HUVECs cultured with 10% heat-inactivated serum from healthy children. HSP: HUVECs cultured with 10% heat-inactivated serum from HSP patients. HSP + MSC: HSP-conditioned HUVECs co-cultured with hUC-MSCs. HSP+MSC+TFA: HUVECs pretreated with colivelin TFA before HSP-conditioned and co-culture. Data are mean ± SEM, n = 3 independent experiments. *** P < 0.001.

Exposure of HUVECs to HSP serum resulted in a marked increase in IL6 and STAT3 mRNA expression (Figure 3B–C), accompanied by elevated protein levels of IL-6, total STAT3, and phosphorylated STAT3 (p-STAT3) (Figure 3D–G), compared with control serum–treated cells. Co-culture with hUC-MSCs significantly reduced IL-6 and p-STAT3 protein levels in HSP-stimulated HUVECs, while total STAT3 expression was only modestly affected.

Figure 3.

hUC-MSCs inhibit STAT3 activation in HSP-stimulated endothelial and immune cells. (A) CCK-8 assay assessing colivelin TFA cytotoxicity in HUVECs, identifying 5.0 μM as a non-cytotoxic working concentration. (B-C) Relative mRNA expression of IL6 and STAT3 in HUVECs exposed to control serum (Ctrl), HSP serum (HSP), HSP serum co-cultured with hUC-MSCs (HSP+MSC), or HSP serum co-cultured with hUC-MSCs and colivelin TFA (HSP+MSC+TFA). (D) Western blot analysis of IL-6, STAT3, and phosphorylated STAT3 (p-STAT3) in HUVECs; GAPDH served as a loading control. (E-G) Quantification of corresponding protein levels. (H) Western blots analysis of IL-6, STAT3, and p-STAT3 in CD4+ T cells under the indicated treatment conditions. (I-J) Quantification of protein expression in CD4+ T cells. (L) Western blot analysis of IL-6, STAT3, and p-STAT3 in hUC-MSCs cultured with Ctrl serum, HSP serum, or HSP serum plus colivelin TFA. (M-O) Quantification of protein expression in hUC-MSCs. Data are mean ± SEM; n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

To assess the effect of pharmacological STAT3 modulation, colivelin TFA was applied as a STAT3 agonist. CCK-8 assays showed that colivelin TFA at 5.0 μM did not affect HUVEC viability and was therefore used for subsequent experiments (Figure 3A). Under these conditions, colivelin TFA treatment increased IL-6 and p-STAT3 expression in HSP serum–stimulated HUVECs co-cultured with hUC-MSCs, restoring protein levels comparable to those observed in HSP-treated cells (Figure 3B–G).

Similar expression patterns were observed in CD4⁺ T cells. HSP stimulation increased IL-6, total STAT3, and p-STAT3 protein levels, whereas co-culture with hUC-MSCs reduced the abundance of these proteins (Figure 3H–J). Addition of colivelin TFA reversed the hUC-MSC–associated reductions in IL-6 and p-STAT3 expression in CD4⁺ T cells.

In hUC-MSCs, exposure to HSP serum increased IL-6 protein expression, while no significant changes were detected in total STAT3 or p-STAT3 levels (Figure 3L–O). Treatment with colivelin TFA further increased IL-6 and p-STAT3 expression in hUC-MSCs without altering STAT3 abundance.

To investigate the therapeutic potential of hUC-MSCs in vivo, the authors established an ovalbumin (OVA) induced HSP rat model. Compared with controls (Supplementary 1B), HSP rats developed widespread purpuric lesions (Supplementary 1C) and histological evidence of multi-organ injury, including renal mesangial proliferation, joint edema, and intestinal villous atrophy (Figure 4). Systemic administration of hUC-MSCs markedly alleviated these abnormalities, reducing skin lesions (Supplementary 1D) and restoring renal, joint, and intestinal architecture (Figure 4). In contrast, co-treatment with colivelin TFA abrogated the therapeutic effects of hUC-MSCs, with rats showing persistent skin lesions, lymphadenopathy, and severe tissue pathology.

Figure 4.

hUC-MSCs alleviate systemic pathology in an OVA-induced rat model of HSP. H&E staining of kidney, ankle joint, and intestine. HSP rats showed glomerular edema (red arrow), joint space narrowing (red circle), and villous atrophy (red rectangle and arrow). hUC-MSCs ameliorated these changes, while colivelin TFA abrogated the therapeutic effects and induced synovitis. n = 10 rats per group, Scale bar = 100 μm. Ctrl: healthy control. HSP: HSP model. HSP+MSC: hUC-MSCs treated HSP model. HSP+MSC+TFA: hUC-MSCs + colivelin TFA treated HSP model.

Flow cytometric analysis of splenic CD4+ T cells revealed that HSP rats exhibited increased Th17 cells, reduced Tregs, and a significantly elevated Th17/Treg ratio (Figure 5A–C). hUC-MSCs treatment reversed this imbalance, suppressing Th17 expansion and enhancing Treg accumulation. However, STAT3 reactivation with colivelin TFA negated these effects, reinstating a Th17-dominant phenotype.

Figure 5.

hUC-MSCs restore Th17/Treg balance in HSP rats through STAT3 inhibition. Flow cytometric analysis of splenic CD4+ T cells. (A) Gating strategy for Th17 (CD4+IL-17A+) and Treg (CD4+Foxp3+) subsets. (B) Percentages of Th17 and Treg cells. (C) Th17/Treg ratio across groups: healthy control (Ctrl), HSP model (HSP), hUC-MSCs treated (HSP+MSC), and hUC-MSCs + colivelin TFA treated (HSP+MSC+TFA). hUC-MSCs normalized Th17/Treg balance, whereas colivelin TFA negated this effect. n = 10 rats per group, * P < 0.05, ** P < 0.01, *** P < 0.001.

Western blot analysis of splenic tissues showed that HSP rats had increased expression of IL-6, STAT3, p-STAT3, and RORγt compared with controls (Figure 6A–E). These changes were accompanied by an expansion of Th17 cells and reduction of Tregs (Figure 6F–G). hUC-MSCs treatment significantly attenuated STAT3 pathway activation, suppressed RORγt, and restored Th17/Treg balance. In contrast, colivelin TFA reactivation reversed these effects, re-establishing Th17 predominance.

Figure 6.

hUC-MSCs downregulate STAT3 signaling and re-establish Th17/Treg homeostasis. (A)Western blot of IL-6, STAT3, p-STAT3, and RORγt in spleen tissues from Ctrl, HSP, HSP+MSC, and HSP+MSC+TFA groups. (B–E) Quantification of IL-6, RORγt, STAT3, and p-STAT3 expression. (F–G) Relative mRNA expression of Th17- and Treg-associated markers. hUC-MSCs suppressed STAT3 activation and RORγt expression, reducing Th17 expansion and partially restoring Treg levels. Colivelin TFA reversed these effects. Data are mean ± SEM, n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.