The potency of aloe emodin‐loaded nanoparticles in conjunction with IFN‐γ for the pretreatment of mesenchymal stem cells with class II transactivator silence to alleviate severe acute pancreatitis

Abstract

Mesenchymal stem cells (MSCs) have a moderate impact on the therapy of severe acute pancreatitis. This study seeks to improve the therapeutic effectiveness of MSCs. By preconditioning them via the upregulation of critical anti‐inflammatory molecules, so diminishing immune rejection, we are creating a path for more effective treatments. Aloe emodin (AE), a natural active monomer with low‐toxicity, in conjunction with interferon gamma (IFN‐γ) (I‐AE), markedly upregulated immunosuppressive molecules indoleamine 2,3‐dioxygenase and programmed cell death‐Ligand 1 in MSCs, thereby pharmacologically modulating the inhibition of CD4 − T cell activation in vitro effectively. Transient transfection of small interfering RNA silenced the class II transactivator (CIITA) gene expression of umbilical cord mesenchymal stem cells (UMSCs) interfering with human leukocyte antigen class II expression to avert immune rejection. AE‐loaded nanoparticles efficiently maintained proliferation inhibition of MSCs within a manageable range by sustained release. UMSCs pretreated by I‐AE with CIITA silencing preserved pancreatic structure as evidenced by diminished acinar cell death, reduced pancreatic edema and inflammation, and significantly lowered serum amylase levels The encouraging potential of UMSCs with CIITA gene silencing combined with AE and IFN‐γ pretreatment offers optimism for clinical application in pancreatitis therapy.

INTRODUCTION

Severe acute pancreatitis (SAP) is a formidable severe illness characterized by systemic and localized consequences.^1,2 Notwithstanding recent advancements in surgical intervention, the therapeutic efficacy of SAP has not markedly improved, and the overall mortality rate persists at roughly 17%. The current clinical management for SAP primarily aims to alleviate symptoms through fluid resuscitation, early countershock, acidosis correction, administration of pancreatic enzyme inhibitors, parenteral nutritional support, infection prevention, surgical intervention, and Traditional Chinese Medicine (TCM).^3–8 Nonetheless, given the significant clinical mortality rate linked to SAP, there is an imperative necessity for the formulation of more efficacious treatment options.

Mesenchymal stem cells (MSCs) are a category of stem cells characterized by their ability for self-renewal and multidirectional differentiation. MSCs modulate the inflammatory signaling axis to produce their anti-inflammatory actions. This effect is accomplished by the synthesis of anti-inflammatory molecules, growth factors, exosomes, and chemokines, along with direct interaction between MSCs and immune cells.⁹ The principal regulatory molecules synthesized by MSCs comprise PGE2, TSG-6, indoleamine 2,3-dioxygenase (IDO), iNOS, TGF-β, HO1, programmed cell death-Ligand 1 (PD-L1), and so forth.^10,11 The research in the domain of MSCs has led to expedited advancement in their therapeutic application. The advancement of novel therapies and personalized medications utilizing MSCs is on the rise, demonstrating efficacy in the management of various conditions, including myocardial infarction, stroke, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Crohn's disease, arthritis, and so forth.^12–15

MSCs have considerable plasticity in modulating inflammation.¹⁶ In the absence of stimulus, they do not demonstrate immune regulatory effects. Immune regulatory molecules are considerably increased in response to inflammatory mediators, hence exhibiting anti-inflammatory actions.^17,18 To date, only type II interferon gamma (IFN-γ) has been recognized as a promising pharmacological agent for the pretreatment of MSCs in clinical investigations for disease management.¹⁹ Our prior research indicated that MSCs preconditioned with a combination of chloroquine/tamoxifen and IFN-γ exhibited more efficiency in the intervention of AP than MSCs preconditioned with IFN-γ alone. This was ascribed to the significantly heightened expression of PD-L1, iNOS, and IDO in the former. Nonetheless, it was also shown that the two medications impeded the multiplication of MSCs, hence constraining the therapeutic application of the treatment. To identify efficient and low-toxicity medicines for the pretreatment of MSCs, natural active monomers from TCM were evaluated. Chaiqin Chengqi Decoction (CQCQD) is a clinically proven TCM recipe for the treatment of SAP, with rhubarb serving as the principal drug.^20,21 As a result, further investigation was conducted into active monomers. The preliminary experiment indicated that both emodin and aloe emodin (AE) functioned in a synergistic manner with IFN-γ in upregulating the immunosuppressive molecules of MSCs. The objective is to investigate the regulatory effects of processed MSCs in vivo and in vitro, and to explore the latent mechanisms of AE and IFN-γ.

The study of stem cells has seen substantial advancements in clinical translation in recent years. In August 2020, Stempeitics Research Pvt. Ltd. obtained regulatory approval from the Drug Controller General of India to introduce Stempeucel in India for the treatment of critical limb ischemia resulting from severe arteriosclerosis or Buerger's disease. As of now, several stem cell therapies have received global clinical approval, with more than 60% employing MSCs. Moreover, about 2000 clinical trials are presently in progress. Stem cell therapy has proven to be an effective treatment for various diseases, including cardiovascular, orthopedic, obstetric and gynecological, neurological, and respiratory ailments. The precise sorts of disorders encompass several dozen, including acute myocardial infarction, arthritis, stroke, ovarian dysfunction, chronic obstructive pulmonary disease, and so forth. This is expected to promote the development of novel strategies for SAP treatment, thereby remedying the existing shortage of effective therapeutic alternatives for this condition.

RESULTS

Isolation and characterization of umbilical cord mesenchymal stem cells (UMSCs)

In this investigation, UMSCs were isolated from healthy umbilical cords by an explant technique. The migration and subsequent proliferation of the dispersed spindle-shaped cells from the explant to the bottom of the dish within 1 week. After an additional 3 days, many cell colonies became apparent at the bottom of the dish. After around 14 days, a significant number of cell colonies merge. At this stage, the cells can be passaged for the first time, demonstrating a significant rise in proliferation rate (Figure 1A). UMSCs exhibit the defining hallmarks of MSCs, such as CD105 and CD73, while lacking CD34 (Figure 1B). Moreover, the cells demonstrated the capacity for differentiation into adipose, osteogenic, and chondrogenic lineages (Figure 1C).

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Upregulation of pivotal immunosuppressive mediators in UMSCs by AE–IFN-γ

IDO and PD-L1 are regarded as the foremost immunosuppressive molecules in MSCs, as demonstrated by prior studies.^22,23 IFN-γ has been shown to stimulate the production of IDO in antigen-presenting cells, facilitating the conversion of tryptophan to kynurenine. This mechanism suppresses T cell-mediated immunological responses.^24,25 PD-L1, a member of the B7 family, can be activated by IFN-γ in antigen-presenting cells and CD3+ cells.²⁶ This overexpression will influence T cell activation and tolerance via interaction with T cell receptors.^27,28

The mRNA expression of various immunoregulatory molecules in UMSCs was evaluated after pretreatment with AE and IFN-γ, referred to as I-AE. The expression of IDO and PD-L1 was notably enhanced by I-AE (Figure 2A). To attain the maximal efficacy of pharmacotherapy, the duration of treatment and drug concentration were evaluated. The expression of IDO and PD-L1 demonstrated a much greater rise after a 24-h treatment period compared with other treatment durations (Figure 2B). Of the four drug concentrations used for pretreatment, the AE concentration of 20 µg/mL exhibited the most pronounced elevation in IDO and PD-L1 levels (Figure 2C). At elevated drug doses, the expression of the two molecules did not increase further, signifying that saturation had been attained. An immunoblotting test was performed for semiquantiative analysis of protein expression, revealing that IDO was not constitutively expressed in UMSCs. The results indicated that I-AE significantly increased protein expression relative to IFN-γ treatment alone (Figure 2D), which was consistent with the quantitative polymerase chain reaction (qPCR) findings.

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In contrast, PD-L1 is consistently expressed on the membrane of UMSCs, rather than in the cytoplasm.^23,27 The protein's presence of the cell surface was validated using flow cytometry (Figure 2F), however it could not be ascertained via immunoblotting in the cell lysate of UMSCs (Figure 2E).

In the cytotoxicity assay, the cell viability shown a progressive decrease with increasing AE concentration. Nonetheless, all rates above 70%. The drug concentration utilized in the previously described tests was 20 µg/mL, achieving a permissible cell inhibition rate of 70% (Figure 2H). It was noted that AE exhibits minimal toxicity to UMSCs and does not induce cell detachment or apoptosis, as observed microscopically. This is likely attributed to the proliferative inhibition of AE on UMSCs.

In summary, the conjunction of AE and IFN-γ markedly enhanced the expression of immunosuppressive molecules, specifically IDO and PD-L1, in UMSCs with minimal toxicity.

Preparation and characterization of AE-loaded nanoparticles (AE NPs)

A preliminary investigation demonstrated that poly(lactic-co-glycolic acid) (PLGA) NPs are a more effective encapsulating carrier for AE than liposomes. The efficacy of AE encapsulation was assessed at 520 nm in a 10% ammonia solution using ultraviolet (UV) spectrophotometry (Figure 3A). A series of standard solutions with differing concentrations were generated, and their UV absorbance at 520 nm was measured to construct the standard curve (Figure 3B). During the evaluation of AE NPs prescriptions, it was noted that the encapsulation efficiency of AE significantly increased when the polyvinyl alcohol (PVA) content rose from 0.5% to 2% (Figure 3D). In the prescription, PVA functioned as the surfactant in the formulation, creating droplets that enclose PLGA, within which the active component AE is contained. An increase in PVA concentration led to improved stability of emulsion droplets, inhibiting AE leakage during emulsification and hence enhancing encapsulation efficiency. The mean particle size of the optimized AE NPs was determined to be 126.27 ± 5.02 nm, with an average zeta potential of −36 ± 0.93 mV (Figure 3E). The findings demonstrated that the AE NPs were spherical, nanoscale in size, and exhibited excellent dispersibility and homogeneity, hence validating the successful synthesis of the NPs (Figure 3F). Moreover, the particle size was determined to be comparable to that acquired from NP tracking analyzer (NTA) detection. In comparison to the free drug, the NPs exhibited significant sustained release characteristics as previously mentioned (Figure 3C). Considering the continuous release profile of the NPs, AE NPs were formulated to stimulate UMSCs, since the internalized NPs would persist in enhancing the cells postadministration in vivo.^29,30 The decrease in cumulative drug release was likely due to the presence of sodium azide in the release medium, as AE was prone to drug degradation after oxidation.

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A comparison of the mRNA levels of IDO and PD-L1 in UMSCs after stimulation by IFN-γ in conjunction with either free AE or AE encapsulated in NPs, demonstrated that the boosting effect was comparable in both formulations. This indicates that nanoencapsulation preserved the drug's efficacy (Figure 3G). The toxicity experiment revealed that the inhibitory effect of AE NPs on UMSCs was markedly reduced compared with the free medication at doses under 80 µg/mL. It may be inferred that the encapsulation of the drug into NPs has enhanced drug tolerance towards UMSCs (Figure 3H). The cellular absorption of AE NPs was examined (Figure 3I). Cellular uptake of C6-labeled NPs was noted within 0.5 h. During the early stages (0.5 and 1 h), the quantity of lysosomes (shown by red fluorescence) was comparatively minimal. After a 2-h incubation period, a significant rise in the quantity of NPs colocalized with lysosomes was noted, as evidenced by the presence of yellow dots. After 4 h, the diminished green fluorescence signified a progressive breakdown of the NPs within the lysosomes. The endocytosis of the AE NPs in MSCs was highly efficient. In conclusion, AE NPs shown the ability to mitigate the inhibitory impact on cell growth within a specific concentration range. The forthcoming experiment will investigate the impact of NPs in vivo.

Characterization of UMSCs following small interfering RNA (siRNA) transfection

In an initial in vivo trial, the combination of AE with IFN-γ for the pretreatment of UMSCs (I-AE UMSCs) proved less successful than the IFN-γ preprocessed UMSCs group (I-UMSCs) in mitigating inflammation (data not shown). Literature indicates that IFN-γ induces the production of human leukocyte antigen class I (HLA-I) and HLA-II class molecules in UMSCs.³¹ HLA-II predominantly facilitates immunological rejection in CD4 + T cells.^32–34 It might be assumed that the combination of AE and IFN-γ led to a more significant elevation of HLA-II molecules, thereby eliciting a stronger immunological rejection response. This may explain the reduced treatment efficacy noted in the I-AE UMSCs group.

The expression of the HLA-II gene is rigorously regulated by the RFX complex which consists of RFXAP, RFX5, and RFXANK subunits, as well as class II transactivator (CIITA).^35,36 A lot of research has shown that the targeted deletion of transcription factors crucial for HLA-II gene expression can significantly impair HLA-II gene expression. Patients with naked lymphocyte syndrome exhibit a deficiency in HLA-II gene expression due to mutations in one of the four distinct transcription factor genes (CIITA, RFX5, RFXAP, or RFXANK).³⁷ A mouse model deficient in HLA-II has been created through the knockout of CIITA.³⁸ The targeted deletion of CIITA or RFXANK in human pluripotent stem cells leads to the lack of HLA-II expression in the differentiated offspring of these cells.^39,40 Due to the growth characteristics and the restricted number of generations of UMSCs produced in vitro, transient transfection of siRNA was utilized to inhibit CIITA gene expression in UMSCs.

The aim was to assess the variations in CIITA expression in UMSCs following various pretreatments. The findings indicated that the I-AE group showed a more significant elevation of CIITA expression compared with the IFN-γ group. Furthermore, the ascending correlation between CIITA and IDO was analogous (Figure 4C). The primer sequence is presented in Table S1.

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Initially, the ideal quantity of transfection reagent (polyethylenimine [PEI]) was determined using fluorescent FAM-RNA. The transfection effectiveness of siRNA attained 82% as determined by flow cytometry detection when PEI was utilized at a 1:1.5 (m/m) ratio (Figure 4A). The assessment of siRNA quantity revealed that 0.5 μg was the optimal dosage (Figure 4B). The expression level of CIITA shown a significant reduction relative to the transfected negative group, so confirming the successful silencing of the CIITA gene in UMSCs (Figure 3D). The CIITA expression level in the transfected negative control group demonstrated a reduction relative to the no treatment group (NT), likely due to the inhibitory effects of transfection reagents on cellular proliferation.

Modulating the immunosuppressive effect of UMSCs with AE–IFN-γ in vitro

Literature indicates that both IDO and PD-L1 exert inhibitory effects on CD4 + T cell activation. IDO catalyzes the conversion of tryptophan, an important amino acid, into different metabolites, resulting in tryptophan consumption. The crucial role of tryptophan in T cell function means that its loss can cause a transition in cellular metabolic pathways from glycolysis to oxidative phosphorylation, ultimately resulting in T cell malfunction.^24,41,42 Consequently, the PD-L1 generated by MSCs can interact with PD-1 of CD4 + T cells, thereby diminishing their IL-2 release and facilitating T cell immunological suppression.^23,43

CD4 + T cells were isolated from murine splenic tissue by flow cytometry sorting. The percentage of CD4 + T cells in a mouse spleen cell was roughly 15%, and the quality of the isolated CD4 + T cells was around 95% (Figure 5B), meeting the requirements for the following tests. Before coculturing CD4 + T cells with UMSCs, the percentage of activated CD4 + T cells (CD69+) induced by CD3/28 was roughly 33%. After coculturing with UMSCs, a decrease in the fraction of activated CD4 + T cells was noted. Conversely, the pretreatment UMSCs group exhibited enhanced efficacy in activating CD4 + T cells, with the combination of AE and IFN-γ treatment producing the most significant effect (Figure 5A). The results indicate that the amalgamation of AE with IFN-γ produced the most efficacious activation of UMSCs.

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As previously described in Section 2.2, the combination of AE and IFN-γ significantly increased IDO expression in UMSCs, as seen by heightened mRNA and protein levels. To clarify the specific signaling mechanism by which AE augmented IDO expression, changes in the activation status of the signaling pathway were observed. The findings indicated a negative connection between extracellular signal-regulated kinase (Erk) phosphorylation (p-ERK) and IDO (Figure 5C). It was hypothesized that AE may augment IDO expression by disrupting p-ERK formation. Validation was performed utilizing the p-ERK inhibitor PD98059. PD98059 was found to diminish IDO expression, aligning with the results of a prior investigation (Figure 5D). The findings demonstrated that AE's modulation of IDO expression was not linked to p-ERK.

The JAK-STAT signaling pathway is a fundamental mechanism that facilitates interferon activation,⁴⁴ prompting an investigation into the effects of AE on this system. The findings revealed a significant association between changes in STAT1 phosphorylation and IDO expression after AE stimulation (Figure 5E). This indicates that AE positively regulates the immunosuppressive ability of UMSCs by directly enhancing the traditional interferon pathway, particularly the p-STAT1-IDO axis.

I-AE in conjunction with CIITA suppressed UMSCs for in vivo intervention of SAP

The treatment effect of the combination of I-AE and CIITA silenced UMSCs was significantly greater than that of the CIITA silenced UMSCs group alone, demonstrating that CIITA silencing substantially mitigated immunological rejection associated with UMSCs. The I-AE UMSCs shown effectiveness in protecting pancreatic architecture, as indicated by a decrease in acinar cell death, mitigation of pancreatic edema and inflammation, and a significant reduction in serum amylase levels (Figure 6A–C).

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Compared with the SAP group, both the I-UMSCs group and the I-AE UMSCs group exhibited the ability to reduce the inflammatory factor TNF-α in the lung lavage fluid, with the latter group showing a more significant decrease (Figure 6F). Hematoxylin and eosin (H&E) staining of lung tissue demonstrated that I-AE UMSCs were notably effective in mitigating pulmonary edema and inflammation (Figure 6D). To evaluate the effect of UMSCs on immune cells in vivo, immunofluorescence labeling of pancreatic tissue was performed (Figure S1). The infiltration of CD4 + T lymphocytes in the lungs of the SAP group was markedly greater than that found in the I-UMSCs and I-AE UMSCs groups. This discovery aligns with other research indicating that UMSCs can affect the function of CD4 + T cells.

The results shown that UMSCs with CIITA gene silencing, in conjunction with AE and IFN-γ pretreatment, significantly reduced the progression of SAP in mice, indicating potential for SAP therapy.

DISCUSSION

It is important to acknowledge that, alongside our research, there are other publications in the literature regarding the application of small molecule immunomodulators for the pretreatment of MSCs to augment their therapeutic efficacy. The combination of UMSCs and tanshinone ⅡA has been explored as a potential therapy for acute lung damage.⁴⁵ Pretreatment of bone marrow MSCs with resveratrol has been shown to efficiently inhibit pancreatic cell death and facilitate the regeneration of damaged vasculature.¹⁷ The emodin and aloe-emodin extracted from rhubarb in this study have been shown to exhibit a remarkable regulatory role in the critical signaling pathways of MSCs, particularly in interferon signaling. This is accomplished by promoting interferon-stimulated response elements (gamma-activated sequence [GAS]) and GAS-driven cis-reporting systems.⁴⁶ Rhubarb has been shown to mitigate pancreatic pathological damage by decreasing the concentrations of inflammatory cytokines in the blood and ascites. Furthermore, it has been demonstrated to suppress the secretion of pancreatic enzymes, such as trypsin and pancreatic lipase, while enhancing intestinal peristalsis, alleviating intestinal paralysis, facilitating the excretion of intestinal endotoxins, improving microcirculation, and preventing microthrombus formation.^47–49 Emodin and AE, the main compounds in rhubarb, have shown significant clinical effectiveness in the management of acute pancreatitis. Thus, the project's design effectively addresses two critical challenges concurrently, rendering it a highly viable option for clinical application.

Emodin and AE were chosen for further study because of their high concentration among the pharmacologically active constituents of rhubarb, thereby ensuring their availability.⁵⁰ The initial analysis indicated that both drugs enhanced the expression of molecules, including IDO, PD-L1, and iNOS, at both protein and RNA levels. Nonetheless, the impact of AE was more significant than that of emodin. Consequently, AE was chosen for additional examination. The variation in their function can be ascribed to the singular structural difference, specifically the methyl group at the 6-position of the anthraquinone ring, leading to a divergence in affinity for the target.

The variation in encapsulation efficiency of AE across different vectors was examined. The anthraquinone ring of AE demonstrates hydrophobicity instead of hydrophilicity, leading to inadequate water solubility and very limited encapsulation effectiveness in amphiphilic liposome materials. This is attributable to the existence of phenolic hydroxyl and hydroxymethyl groups within the ring. The initial investigations indicated that the encapsulation effectiveness of AE within liposomes was below 20%. Upon the application of heat, the chemical can be solubilized in chloroform and methanol, indicating a degree of lipophilicity. Consequently, PLGA with greater lipophilicity compared with phospholipids was utilized. Subsequent to the prescription optimization process, AE-loaded PLGA NPs demonstrate elevated encapsulation efficiency. Certain classic anthraquinone pharmaceuticals, like, doxorubicin hydrochloride and mitoxantrone hydrochloride, have attained maximal encapsulation efficiency in liposomes, attributable to the amphiphilicity induced by the salinization of the basic groups within the anthraquinone structure. Nevertheless, the composition of AE was devoid of any salt-forming groups, making the liposomes ineffective as vectors.

This study has inherent limitations. The chosen carrier material, PLGA, has not received approval for the pretreatment of MSCs in vitro. However, it may be reasonably assumed that the standards for in vitro use will be considerably less rigorous than those applicable to intravenous administration. Second, execution of the proposed MSC pretreatment scheme will extend the period of in vitro cell culturing, hence increasing the cell therapy cycle and escalating expenses. Third, Given the presence of Dahuang derivatives in CQCQD, which has already been clinically available for the treatment of acute pancreatitis, it may be hypothesized that IFN-γ, MSCs, and the Decoction could be administered simultaneously to achieve synergistic effects, thus avoiding the necessity for cumbersome in vitro pretreatment. The present study has yet to investigate this issue.

CONCLUSION

The pressing clinical demand for SAP treatment and the imperative for clinical translation have necessitated the formulation of a strategy based on MSC therapy. Clinically authorized natural active monomers were utilized alongside IFN-γ to precondition MSCs, aiming to enhance critical regulatory components within the interferon signaling cascade. siRNA-mediated gene silencing was utilized to downregulate HLA-II expression in MSCs, aiming to diminish immune rejection. The inclusion of pharmacological regulation and nanotechnology significantly improved the effectiveness of MSC-based therapy. This technique led to a significant expression increase in the protein level of immunosuppressive IDO and PD-L1 of MSCs. Furthermore, it resulted in a notable suppression of CD4 + T cell activation in vitro, which subsequently protected the pancreatic structure by reducing acinar cell apoptosis, pancreatic edema, and inflammation. The integration of UMSCs with CIITA gene silencing, together with AE and IFN-γ pretreatment, signifies a viable pathway for future therapeutic application.

MATERIALS AND METHODS

Assessment of the immune regulator expression in UMSCs poststimulation

UMSCs were inoculated in six-well plates and incubated for one night. The culture medium was subsequently substituted with 2 mL of complete culture medium with different concentrations of AE (Chengdu Desite Biotechnology) and AE NPs (5 ng/mL IFN-γ). The untreated cells served as the control group. The media were meticulously combined and then positioned in the incubator for cultivation. The expression of MSC immune regulators, including PD-L1, IDO, TSG-6, TGF-β1, HO-1, and IL-10, was assessed by real-time qPCR (RT-qPCR). The pertinent primer sequences are included in supplemental materials (Table S1). Western blot analysis was utilized to assess the protein expression of IDO and PD-L1.

Preparation and characterization of AE NPs

• (1)

  Preparation of AE NPs

  AE NPs were synthesized using the emulsion/solvent evaporation technique. A solution of PLGA at a concentration of 80 mg/mL in chloroform/methanol solution (4:1, v/v) containing AE as the inner oil phase was emulsified with a 1% PVA solution as the outer aqueous phase through ultrasonication for 5 min, yielding an oil-in-water (O/W) emulsion. Thereafter, the organic phase was evaporated under reduced pressure at 37°C, yielding a colloidal suspension of PLGA NPs. Subsequently, the unencapsulated free AE in the precipitates obtained from centrifugation at 2000 rpm was eliminated. The AE NPs were obtained using ultracentrifugation (13,300 rpm, 90 min, 4°C) and subsequently resuspended in normal saline at 4°C before use.

• (2)

  Drug entrapment efficiency (EE)

  The drug EE, represented as a percentage, was determined by dividing the quantity of drug entrapped by the quantity of drug added and multiplying the result by 100. Thereafter, the amount of AE was quantified using UV spectrophotometry.

  The parameters of the AE NP formulation, such as PLGA amount, PVA concentration, and ultrasonication duration, were adjusted by a single-factor screening method, utilizing EE, and particle size as metrics.

• (3)

  Measurement of particle size and zeta potential of AE NPs

  The NP size distribution and zeta potential were assessed using an NTA.

• (4)

  Transmission electron microscopy (TEM)

  The NPs' morphology was seen via TEM (H-600, Hitachi) after negative staining with a 2% phosphotungstic acid solution.

• (5)

  Intracellular uptake of NPs in vitro

UMSCs were seeded in 24-well plates at a density of 5 × 10⁴ cells per well. After a 24-h incubation period, the medium was substituted with fresh medium containing C6-labeled AE NPs at a final concentration of 25 ng/mL. After incubating with UMSCs for 0.5, 1, 2, and 4 h, the media were discarded, and lysosomal staining was conducted utilizing Lyso-Tracker (Beyotime Biotechnology). Thereafter, the medium was eliminated, and the cells were rinsed three times with cold phosphate-buffered saline (PBS). The nuclei were then counterstained with 4′,6-diamidino-2-phenylindole. The cells were subsequently viewed and imaged utilizing an Axio Imager A2 fluorescence microscope (Zeiss).

Assessment of in vitro drug release

The in vitro drug release of AE NPs was examined using the dialysis bag method at 37°C. A dialysis bag (Biotopped) with a molecular weight cut-off size of 3500 Da was employed, and PBS (pH 7.4) containing 0.2% (w/v) sodium dodecyl sulfate and 0.01% sodium azide served as the release medium. In conclusion, 1 mL of AE NPs dispersion was placed into the dialysis bag, with both ends fastened by threads. The dialysis bag was submerged in 50 mL of the dissolving liquid and stirred at 100 rpm at 37°C. At specified time intervals, 1 mL of the dissolving media was extracted and substituted with an equal volume of the new medium. The drug concentration in each sample was analyzed using UV spectrophotometry, and the cumulative percentage of AE released was subsequently estimated.

Transfection of UMSCs with si-CIITA

• (1)

  Screening of transfection conditions using fluorescent N-(3-fluoranthyl)maleimide (FAM) si-RNA

  First, UMSCs were plated in a 24-well plate at 24 h before transfection achieving a confluence of 50%–70%. Before transfection, 0.5 μm of FAM si-RNA was mixed with different amounts of the transfection reagent PEI (Table S2) in DMEM and permitted to incubate at room temperature for 5 min. Subsequently, the planted cells were rinsed thrice with sterile PBS, following which the preceding medium was discarded. Subsequently, 200 µL of fresh DMEM incorporating the previously described siRNA–PEI mixture was introduced. After a 4–6-h cultivation period, the cells were collected, and the transfection effectiveness was assessed by flow cytometry. Furthermore, the transfected cells were observed using a fluorescent microscope.

• (2)

  Transfection of UMSCs with CIITA siRNA

UMSCs were seeded in a 12-well plate at an optimal density of 50%–70% for overnight incubation. Transfection was subsequently performed using 0.5 μg of CIITA siRNA or a negative control siRNA with PEI (Sigma). After a 5-h transfection phase, the medium was substituted with one containing IFN-γ, deviod of antibiotics, and cultured for an additional 24 h. To verify the knockdown of CIITA, mRNA levels were measured by RT-qPCR, with the primer sequence provided in the Supporting Information materials (Table S3).

Animal model and treatment

Male C57BL/6 mice, aged 6–8 weeks, were bred at the Experimental Animal Centre of Sichuan University (Chengdu, China). All animal tests performed in this work received approval from the Animal Care and Use Committee of Sichuan University (20190509023, Chengdu, Sichuan, China).

Mice were injected caerulein (CAE, Yuanye) (75 μg/kg) intraperitoneally in 10 consecutive doses, with a 1-h gap between each injection, to establish a model of SAP. Lipopolysaccharide (LPS) (15 mg/kg) was delivered intraperitoneally immediately after the last cerulein injection. The UMSCs were administered gradually by the tail vein immediately after LPS injection, at a dosage of 1 × 10⁶ cells/kg, over 1 h in the treatment groups. Consequently, the animals were euthanized 24 h post-LPS injection to obtain lung lavage fluid and other tissues.

H&E staining

The pancreatic and lungs tissue specimens were fixed in 10% (w/v) phosphate-buffered formalin and embedded in paraffin following standard histological protocols. Sections of 5 μm in thickness from each sample were stained with H&E. The stained sections were examined using a light microscope (OLYMPUS BX43), and the generated pictures were analyzed to detect any histological changes. A double-blind methodology was utilized to solicit pathologists for pathological scoring of the pancreas and lungs, respectively. The pancreatic tissue scoring standard table (refer to Table S4) was derived from the scoring criteria established by Kusske,⁵¹ whilst the lung tissue scoring standard table (refer to Table S5) was based on the scoring criteria given by Osman.⁵²

Statistical analysis

All data were shown as means ± standard deviation (n = 3 per group, unless otherwise stated). The disparities between the two groups were evaluated using an unpaired Student's t test, while multiple comparisons were analyzed using a one-way analysis of variance followed by Tukey's multiple comparisons test. All statistical analyses were conducted utilizing GraphPad Prism (version 9.51, GraphPad Software). A p value below 0.05 was deemed indicative of statistical significance.

AUTHOR CONTRIBUTIONS

Yu Zou is responsible for data curation. Qin Qin supplements experiments during the article revision process. Xiaoshuang Song is responsible for the review and editing of the manuscript. Yuchuan Deng, Simeng Liu, and Huimin Liu undertake the methodology. Mao Wang and Yiran Song are in charge of formal analysis. Dujiang Yang, Huimin Lu, Kun Jiang, and Qian Yao are responsible for writing the original draft. Yu Zheng undertakes the conceptualization. All authors have read and approved the final manuscript.

ACKNOWLEDGMENTS

Natural Science Foundation of Sichuan Province (2024NSFSC0606 and 2024YFHZ0053).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data used to support the findings of this study are available from the corresponding author upon request.

ETHICS STATEMENT

All animal experiments involved in the present study were approved by the Animal Care and Use Committee of Sichuan University (20190509023, Chengdu, Sichuan, China).

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