ISX-9 Promotes KGF Secretion From MSCs to Alleviate ALI Through NGFR-ERK-TAU-β-Catenin Signaling Axis

Abstract

Background

Mesenchymal stem cells (MSCs) have been widely studied to alleviate acute lung injury (ALI) due to their paracrine function. However, the microenvironment of inflammatory outbreaks significantly restricted the factors secreted from MSCs like keratinocyte growth factor (KGF). KGF is a growth factor with tissue-repaired ability. Is there a better therapeutic prospect for MSCs in combination with compounds that promote their paracrine function? Through compound screening, we screened out isoxazole-9 (ISX-9) to promote MSCs derived KGF secretion and investigated the underlying mechanisms of action.

Methods

Compounds that promote KGF secretion were screened by a dual-luciferase reporter gene assay. The TMT isotope labeling quantitative technique was used to detect the differential proteins upon ISX-9 administrated to MSCs. The expressions of NGFR, ERK, TAU, and β-catenin were detected by Western blot. In the ALI model, we measured the inflammatory changes by HE staining, SOD content detection, RT-qPCR, immunofluorescence, etc. The influence of ISX-9 on the residence time of MSCs transplantation was explored by optical in vivo imaging.

Results

We found out that ISX-9 can promote the expression of KGF in MSCs. ISX-9 acted on the membrane receptor protein NGFR, upregulated phosphorylation of downstream signaling proteins ERK and TAU, downregulated phosphorylation of β-catenin, and accelerated β-catenin into the nucleus to further increase the expression of KGF. In the ALI model, combined ISX-9 with MSCs treatments upgraded the expression of KGF in the lung, and enhanced the effect of MSCs in reducing inflammation and repairing lung damage compared with MSCs alone.

Conclusions

ISX-9 facilitated the secretion of KGF from MSCs both in vivo and in vitro. The combination of ISX-9 with MSCs enhanced the paracrine function and anti-inflammatory effect of MSCs compared with MSCs applied alone in ALI. ISX-9 played a contributive role in the transplantation of MSCs for the treatment of ALI.

Graphical Abstract

Graphical Abstract.

Significance Statement.

In this project, by establishing a compound screening cell model, ISX-9 that promotes the paracrine function of mesenchymal stem cells was screened out in the compound library, and it was clarified that ISX-9 acts on the membrane receptor protein NGFR of bone marrow mesenchymal stem cells, regulate the downstream ERK-TAU-β-catenin signaling axis, and further enhance the ability of bone marrow mesenchymal stem cells to secrete KGF. In the in vivo ALI model, for the first time, ISX-9 was combined with bone marrow mesenchymal stem cells in the treatment of acute lung injury, and it was confirmed that ISX-9 promoted bone marrow mesenchymal stem cells to relieve inflame.

Introduction

Since 2019, the incidence of acute respiratory distress syndrome (ARDS) has skyrocketed due to the COVID-19 pandemic. ARDS is a clinical syndrome that develops from acute lung injury (ALI) to acute respiratory failure. ALI is characterized by bilateral pulmonary infiltrates with damage to the lung surface barrier and provokes a severe inflammatory response that ultimately leads to high mortality.¹Clinical studies in recent years have found that mesenchymal stem cells(MSCs)-based cellular therapies can repair the barrier,² reduce pulmonary edema,³ relieve inflammation,⁴ and thereby alleviate the pathological features of ALI.⁵

Mesenchymal stem cells (MSCs), which have immunomodulatory functions and the potential to repair damaged lungs,⁶ are considered candidates for the treatment of ALI. The restorative effect of MSCs is mainly mediated by their paracrine mechanisms.⁷ MSCs increase alveolar clearance and reduce pulmonary edema by secreting keratinocyte growth factor (KGF).⁸ MSCs secrete hepatocyte growth factor (HGF)⁹ to restore endothelial permeability in the lungs and vascular endothelial growth factor (VEGF) to promote angiogenesis.¹⁰ Among these cytokines, KGF is one of the most important factors and is widely used to alleviate a variety of lung diseases.

KGF is a homeostatic factor with epithelial protective and repair potential. Many studies have demonstrated that KGF has shown great potential for the treatment of ALI. KGF induces alveolar type II cell proliferation,¹¹ improves pulmonary edema, and reduces apoptosis in vitro. Endotracheal administration of KGF to patients with ALI promotes repair of damaged lungs, improves lung tissue integrity,¹² and improves patient outcomes. However, the paracrine function of MSCs is limited in the inflammatory environment, so the therapeutic effect of KGF from MSCs in ALI is not significant. How to boost the ability of MSCs to secrete KGF in an inflammatory environment is an urgent problem to be solved.

Most of the traditional cell models used for compound screening are judged by the survival rate of model cells,¹³ but the target and mechanism of compounds are not clarified. If a cell model for compound screening is established with a well-defined gene as the target, most of them target the binding site of a specific transcription factor or receptor in the target gene regulatory sequence, so that the active ingredient that acts only on the target gene can be screened. Therefore, in this study, we proposed to use the promoter of the KGF gene as the screening target to construct a compounds screening cell model of KGF-hcMSCs (placental chorionic mesenchymal stem cells). Combined with the paracrine function of MSCs, the KGF-hcMSCs cell model aims to screen compounds that promote MSCs to paracrine KGF, thereby enhancing the effect of KGF-mediated repair of lung injury.

In this study, we focused on searching for compounds that promote KGF expression via compound screening. Subsequently, the underlying mechanism by which compounds promote MSCs to secrete KGF was studied. Finally, we verified the repair effect of compounds cooperating with MSCs in the ALI model. Our research will provide new perspectives on the clinical application of MSCs.

Materials and Methods

Establishment of the KGF-hcMSCs Cell Model

A luciferase reporter gene recombinant plasmid CV123-KGF-promoter-Luc with the KGF gene promoter was made and transfected with a lentiviral plasmid in 293T cells to make a lentiviral solution (Supplementary Fig. S1a). The cells were then infected with the lentivirus to get KGF-hcMSCs that permanently expressed the KGF promoter.

Compounds Screening

The hcMSCs cells stably expressing the KGF promoter were seeded in 96-well plates at a density of 100 μL of 2 × 10⁴ cells per well. There were 80 compounds added from the drug compound library that were related to stem cell action (compound-specific information: product name, chemical name, molecular formula, CAS number, and brief pharmacological description added in Supplementary Excel 1). Five replicate wells were set for each group, and after 72 hours of action, 100 μL of Dual-Lumi Firefly Fluoresceinase Assay Reagent (Dual-Lumi Dual Fluoresceinase reporter gene assay kit, purchased from Beyoncé) was mixed well, left for 10 minutes at room temperature, and the fluorescence intensity was detected using a multifunctional enzyme marker. Subsequently, 100 μL of Dual-Lumi Sea Kidney Fluorophore Assay Working Solution (Dual-Lumi Fluorophore Reporter Gene Assay Kit, purchased from Beyoncé) was added and mixed well. Encapsulate by incubating for 10 minutes at room temperature and measuring the fluorescence intensity using a versatile enzyme marker. The fluorescence intensities were used and reflected the promoter activity of the KGF gene based on the measurement of the luciferase reporter gene to obtain compounds that target the KGF gene promoter and promote effective expression of the luciferase reporter gene.

Cell Culture

BMSC-GFP (RAT) was purchased from Cyagen Biosciences Inc. (RASMX-01101). BMSCs-GFP were cultured in rat mesenchymal stem cell medium (OriCell, RAXMX-90011) and put in a cell culture incubator at 37 °C with 5% CO_2. Cells fused for 72 hours were 90% fused and passed. The hc-MSCs were cultured as described in the previous study.¹⁴ Cell viability was assessed indirectly by CCK-8 assay.

Reagents

ISX-9 (MCE, HY-12323) was solubilized by DMSO in vitro experiments and by adding 10% DMSO followed by 90% (20% SBE-β-CD in saline) in vivo experiments. ERK inhibitor: U0126-EtOH (IU0160, Solarbio). GSK-3β inhibitor: TWS 119 (Selleck, S1590). MSAB (MCE, HY-120697) is a β-catenin inhibitor.

Animal Models

Thirty-six male Sprague-Dawley (SD) rats that were 6-8 weeks old and weighed between 180 and 220 g were bought from the SLAC Animal Company and fed standard animal food and water. The Institutional Animal Care and Use Committee at Shanghai Jiao Tong University gave the go-ahead for all animal procedures. Based on pharmacological experimental methods, rats were randomly divided into 6 groups: (1) control group (Ctrl; n = 6); (2) ALI group (5 mg/kg LPS, intratracheal drip, ALI; n = 6); (3) ISX-9 alone for ALI group (ISX-9 by intraperitoneal injection for 7 days, tracheal drip 5 mg/kg LPS, ALI-ISX-9; n = 6); (4) MSCs alone for ALI group (MSCs via tail vein for 7 days, tracheal drip 5 mg/kg LPS, ALI-MSC; n = 6); (5) MSCs + ISX-9 combination for ALI group (5 mg/kg LPS, endotracheal drip, ISX-9 via intraperitoneal injection, MSCs via tail vein, ALI-ISX-9-MSCs; n = 6); (6) NGFR knockdown group, shNGFR-MSCs + ISX-9 combination for ALI group. A model of ALI was made by putting a drip of LPS into the trachea. Four hours later, ISX-9 and BMSCs were injected. MSCs (1 × 10⁶, iv) were injected once a day at intervals, and ISX-9 (25 mg/kg, ip) was injected daily for 1 week of treatment. After 7 days, rats were euthanized by intraperitoneal injection of an excess of chloral hydrate. For further analysis, BALF and lung tissue were collected and stored at −80 °C.

Histological Examination

Rat lungs were firmed in 4% formaldehyde, embedded in paraffin, and sliced into 5 μm. The method of HE staining was based on a published study.

Immunofluorescence Staining

Lung tissue that has been preserved in paraffin sections is dewaxed with xylene and then rehydrated in an alcohol solution with a concentration gradient. Blocking in 5% serum after 30 minutes of incubation in 0.3% H₂O₂ and 0.01 M Tris inhibits endogenous peroxidase activity and reduces nonspecific binding. Samples were incubated with KGF antibody (DF13342, Affinity, 1:500) overnight at 4 °C, washed with PBS, and then incubated with CY3 goat anti-rabbit IgG (H + L) (Jackson, 111-095-003) (Red) for 1 hour in a light-proof room at room temperature. Subsequently, the sections were restained with DAPI (blue) and sealed. Immunofluorescence signals were detected using fluorescence microscopy.

Quantitative Reverse Transcription-Polymerase Chain Reaction

Lung tissue RNA was extracted using the Tissue RNA Purification Kit PLUS (EZBioscience, RN001-plus), and the isolated RNA was turned into cDNA using 4 EZScript Reverse Transcription Mix II (EZBioscience, RT2GQ). For real-time quantitative qRT-PCR, a LightCycler 96 real-time PCR system (Roche) and a 2xSYBR Green qPCR Mix kit (EZBioscience) were used. The mRNA expression of GAPDH in lung tissue was detected and analyzed by qRT-PCR. Gene primer sequences for qRT-PCR (Supplementary Table S2) were designed on the Primer3 platform(Primer3 Input). All qRT-PCRs were repeated 3 times, and the expression levels of candidate genes were determined by the 2-ΔΔCT method. Expression levels were normalized to the reference gene GAPDH. Data are expressed as mean ± SD, and GraphPad Prism 8 software was used for a one-way ANOVA. *P < .05, **P < .01.***P < .001.

Western Blotting

To verify the expression of KGF, Ang-1(angiopoietin-1), β-catenin, NGFR, ERK, and other proteins, cellular proteins were extracted from MSCs after exposure to different concentrations of ISX-9. Protein concentrations were determined using the BCA assay (CST, #7780, USA). Proteins were separated on SDS-PAGE gels prior to transfer to PVDF membranes (Life Technologies, USA). PVDF membranes were closed in a fast closure solution for 30 minutes before being incubated overnight with various primary antibodies, including KGF (Abcam, ab131162), angiopoietin 1 (Ang-1, Abcam. ab102015, UK), p-β-catenin (Affinity, DF2989), β-catenin (Affinity, BF8016), TAU (Affinity, AF6141), p-TAU (Abways, CY5657), NGFR (Abcam, ab52987), cleaved caspase-3 (Affinity, AF7022), cleaved caspase-9 (Affinity, AF5240), Bax (Wanleibio, WL03446), GAPDH (Abways,AB0037) were used as controls for total proteins. The membranes were then incubated with HRP secondary antibody (Jackson ImmunoResearch Laboratories, 111-035-003, 115-035-003, China). Blots were detected by the ECL technique (115-035-003, China).

Total Protein Concentration in BALF

Seven days after the BMSCs injection created the ALI model in rats, we took bronchoalveolar lavage fluid (BALF), spun down the supernatant, and used the BCA protein kit (CST, #7780, USA) to measure the total protein concentration.

Wet to Dry Ratio (W/D)

After we euthanized the rats, we took out the right lung, weighed its wet weight with a precision electronic balance, and then put the left lung tissue in a 65 °C oven for 48-72 hours until a constant weight (dry weight) was obtained. Three rats were randomly selected in each group, the W/D ratio was calculated to reflect pulmonary edema, and the mean value and SD value were calculated.

Superoxide Dismutase Content

Right lung tissues from different treatment groups were collected, lung tissue homogenates were made with a homogenizer, and the SOD activity in lung tissues was measured with a SOD activity assay kit (Solarbio, BC0175) at a wavelength of 560 nm using an enzyme standardization instrument.

Bioluminescence Imaging

BMSCs labeled with fluorescent (GFP) were injected into rats through the tail vein, and BMSCs alone were injected compared to the 2 treatment groups in combination with ISX-9 injection at 2, 24, 48, 72, and 96 hours post-injection using an in vivo imaging system (IVIS) (Perkin Elmer, Waltham, MA, USA) for bioluminescence imaging.

Analyses of Statistics

In all experiments, 3 replications were carried out. In the quantitative results, the standard deviation (SD) is multiplied by the average. For comparisons among multiple groups, Dunnett’s post hoc test was applied using GraphPad Prism 8.02 software, and Mann-Whitney’s test was used with a one-way analysis of variance (ANOVA).

Results

Establishing KGF-hcMSCs Cell Model for Screening Compounds and Verifying

Eighty compounds related to stem cells were selected in the Bioactive Compound Library. According to the luciferase activity, 9 candidates for activating KGF expression were screened (Fig. 1a). Compound Isoxazole 9(ISX-9) had the greatest degree of activation of KGF expression, and its luciferase activity increased by 3.18-fold compared with the control group; followed by compound PD 98059, its luciferase activity increased by 2.36-fold; the lowest degree of activation of KGF promoter is BIO-acetoxime, its luciferase activity increased by 1.56-fold (the specific information and calculated P values of the 9 compounds are in Supplementary Table S1). To illustrate the potential of candidate compounds to promote hc-MSCs secrete KGF, KGF mRNA levels were detected after hc-MSCs were treated with 9 compounds (Fig. 1b). Among the 9 compounds, ISX-9 has the best effect on up-grading KGF expression. When the concentration of ISX-9 was 10 μM, the degree of promoting KGF mRNA expression was the most significant. MSCs secrete KGF, Ang-1 to repair the lung barrier. Therefore, the level of Ang-1 mRNA in hc-MSCs was also detected by RT-qPCR. The results showed that the expression of Ang-1 mRNA in hc-MSCs was promoted significantly with ISX-9 treated (Fig. 1c). Based on the analysis of the above results, the 3 compounds ISX-9, cardiogenol C hydrochloride, and CH 223191 simultaneously promoted hc-MSCs secrete KGF and Ang-1. Similarly, the effect of 9 candidate compounds on the cell viability of hc-MSCs was detected by CCK-8 (Supplementary Fig. S1). Western blot verified that ISX-9 promoted the secretion of HC-MSCs KGF and Ang-1 after 24 h.

Figure 1.

KGF-hcMSCs were used to screen compounds and validate. (a) Dual luciferase reporter gene results reflecting the degree of KGF activation. Nine compounds acting on KGF-hcMSCs, respectively. KGF degree of activation = RLU measured by firefly fluorophore enzyme reporter gene/RLU measured by sea renin fluorophore enzyme reporter gene. Experiments were repeated 4 times independently, and compared with blank control, *P < .05, **P < .01, ***P < .001, ****P < .0001 indicate significant differences. (b) ISX-9 significantly promotes KGF expression in hc-MSCs at the mRNA level. Values in the figure are the mean ± SD of 3 independent experiments. Compared with the blank control group without compounds addition, *P < .05, **P < .01. (c) ISX-9 significantly promoted the expression of Ang-1 in hc-MSCs at the mRNA level. Compared with the blank control group without compound addition, *P < .05, **P < .01, ***P < .001. (d) ISX-9 significantly promoted the expression of KGF in BMSCs at the mRNA level. Data for each group of individuals are expressed as means SD and represent the results of at least 3 independent experiments. *P < .05; **P < .01; and ***P < 0.001.

The rat acute lung injury (ALI) model is commonly used to explore the repairing effect of MSCs on ALI. Therefore, we introduced rat bone marrow-derived mesenchymal stem cells (BMSCs) as the experimental cell line. The effects of 9 candidate compounds on the secretion of KGF from BMSCs were also detected by RT-qPCR (Fig. 1d). It turned out that ISX-9, CH 223191, PD 98059, and ID 8 all increased KGF expression in BMSCs at mRNA level. Combined with the results of Fig. 1b, ISX-9 and CH 223191 remarkably upgrade KGF mRNA levels both in hc-MSCs and BMSCs. Therefore, ISX-9 and CH 223191 were chosen as the target compounds after secondary verification.

KGF Expression was Upregulated by ISX-9 in BMSCs

We observed that treatment with ISX-9 or CH223191 increased KGF mRNA, with optimal effects at 5 μM and 25 μM, respectively (Fig. 1d). We next explored the effects of 9 compounds on the viability of BMSCs (Fig. 2a). It revealed that viability of BMSCs was not affected after ISX-9 treatment at 0.01-10 μM. At the same time, the effect of 0.01-10 μM ISX-9 on the apoptosis of BMSCs was detected by Western blot. The results showed that 0-10 μM ISX-9 could inhibit cell apoptosis, reduce the expression of apoptosis protein cleaved-caspase-9, and increase the expression of BCL-2 (Supplementary Fig. S2). However, when the concentration of CH223191 exceeded 10 μM, the cell viability was obviously impaired. Moreover, ISX-9 remarkably increased the KGF protein levels (Fig. 2b). Based on the above verification results, ISX-9 was selected as the final target.

Figure 2.

ISX-9 promotes KGF expression in BMSCs. (a) Effect of 9 compounds on cellular activity after action on BMSCs. Values in the figure are the mean ± SD of 3 independent experiments. Compared with the blank control group without compound addition. (b) Western blot assay to detect the effect of 9 compounds on KGF expression by BMSCs Compounds action concentration and time: ISX-9 (5 μM), cardiogenol C (10 μM), CH 223191 (25μM), PD 98059 (10 μM), GW 788388 (5 μM), ID 8 (10 μM), kenpaullone (10 μM), BIO-acetoxime (1 μM), valproic acid (1 μM). N = 3. (c-f) Western blot to probe the optimal action time and concentration of ISX-9 to promote KGF secretion from BMSCs. Western blot detection of KGF protein expression in BMSCs after 12, 24, 48, and 72 hours of ISX-9 action at concentrations of 0,1.25, 2.5, and 5 μM, respectively. N = 3. (g) RT-qPCR was performed to detect the changes in KGF mRNA levels in BMSCs after the action time of ISX-9 at concentrations of 0,1.25, 2.5, and 5 μM for 12, 24, 48, and72 hours, respectively. N = 3. Compared with the blank control group without compound addition, *P < 0.05, **P < 0.01, ***P < 0.001. (h-k) The value of relative protein expression represented the ratio of targeted protein quantitative value to GAPDH quantitative value in each lane. Data are expressed as the mean ± SD of individuals in each group and represent the results of at least 3 independent experiments. *P < .05; **P < .01.

The optimal effect of ISX-9 was explored by treating BMSCs with concentration and time gradients of ISX-9 (Fig. 2c-f). The results of Western blot illustrated that the expression of KGF increased at 24 hours when BMSCs are treated with ISX-9 at 0.01-5 μM, but there was no remarkable change at 12 hours. Furthermore, 1.25 μM ISX-9 enhanced KGF secretion by BMSCs at 48 hours. However, as the time increased to 72 hours, the expression of KGF gradually decreased, which may be due to the long-term action affecting the secretion function of BMSCs (Fig. 2h-2k). RT-qPCR also confirmed this conclusion (Fig. 2g). In conclusion, ISX-9 was most effective in stimulating BMSCs to secrete KGF and inhibiting apoptosis after 24 hours of treatment with BMSCs.

The Potential Mechanism of ISX-9’s Effect on BMSCs was Predicted by Tandem Mass Tag

The above studies have demonstrated that ISX-9 can stimulate BMSCs to secrete KGF after 24 hours, however, the underlying mechanism of this effect remains unclear. Protein-protein interactions within cells can have significant impacts on various biological processes,¹⁵ such as cell proliferation and paracrine signaling. Our study hypothesized that ISX-9 had a considerable impact on certain proteins in BMSCs, ultimately regulating BMSCs to secrete KGF via protein interaction. To investigate this hypothesis, we conducted TMT labeling and LC-MS/MS analysis on BMSCs treated with ISX-9.

Enrichment analysis using GO and KEGG (Fig. 3a and 3b) revealed that treatment of BMSCs with ISX-9 resulted in the upregulation of proteins involved in microtubule binding, neurotrophin signaling, and pathways related to the repair of lung injuries. Among the top 30 upregulated proteins, we observed significant increases in the expression of nerve growth factor receptor (NGFR) and Mapt (TAU), which are associated with neurotrophin signaling pathways¹⁶ and microtubule binding biological processes¹⁷ (Fig. 3c). When compared to the control, ISX-9 acting BMSCs induced upregulation of 233 genes and downregulation of 134 genes (Fig. 3d and 3e). We suspected that NGFR and Mapt (TAU) may play a crucial role in the mechanism underlying ISX-9-induced BMSCs to secrete KGF. To identify potential connections between proteins, we searched for the top 5 proteins with the closest interaction with NGFR in the STRING database (https://string-db.org/). Mapt and KGF (Fgf7) were also used as central proteins (Fig. 3f). Although the top 5 proteins associated with NGFR were not directly related to Mapt (TAU), Mapt/GSK-3β/β-catenin/KGF(Fgf7) showed interactions in the differential protein results. The interaction between Mapt (TAU)/GSK-3β/β-catenin has also been demonstrated in the study of the neurological disease Alzheimer’s disease (AD), where the study of Mapt (TAU) hyperphosphorylation is accompanied by a decrease in phosphorylation of β-catenin and an increase in nuclear translocation of β-catenin.¹⁸ KGF (Fgf7) has been shown to be a target gene regulated by β-catenin in the cAMP-mediated Wnt/β-catenin pathway.¹⁹ These research analyses suggested that ISX-9 promoted KGF expression possibly through NGFR and Mapt (TAU) and downstream proteins.

Figure 3.

TMT quantitative proteomics analysis of key target proteins of ISX-9 in BMSCs. (a) Bubble plot of top 15 for GO enrichment analysis. (b) KEGG enrichment analysis top 20 bubble plot. x indicates the enrichment score, y-axis is the top 5 term information and top 20 pathway information of BP/CC/MF, respectively. The smaller the enrichment P-value, the greater the significance. (c) Cluster heat map for clustering analysis of differential protein expression levels. (d) Numbers of up and downregulated differential proteins. (e) Volcano plot of differential protein distribution, the horizontal coordinate of the volcano plot is log2(FC), the red point on the right side is the upregulated protein, and the blue on the left side is the downregulated protein. The vertical coordinate is −log10(P-value), and the farther the vertical coordinate value is from the 0 point, the larger the difference is. (f) PPI analysis between NFGR,MAPT (TAU), KGF in STRING database for differential protein interaction network analysis.

ISX-9 Acted on NGFR to Transmit Downstream Signals

Based on the analysis of differential proteins, it was assumed that ISX-9 acted on the NGFR and regulated downstream signals. The change in NGFR after processing BMSCS with ISX-9 was verified using qRT-PCR. The results showed that as the concentration of ISX-9 increased, NGFR mRNA also increased (Fig. 4a). Furthermore, changes in NGFR and downstream proteins were explored by Western blot (Fig. 4b, Supplementary Fig. S3b), indicating that ISX-9 processing increases NGFR and related downstream proteins. To verify the target protein of ISX-9, we knocked down the NGFR gene in BMSCs and observed changes in downstream the protein. The infective effect was observed using a fluorescent microscope (Supplementary Fig. 3Sa). The most efficient sequence shNGFR1 was identified using qRT-PCR (Fig. 4d). Western blot analysis confirmed a remarkable decline in NGFR protein expression (Fig. 4c and 4f), indicating successful construction of shNGFR-BMSCs cell line. BMSCs and shNGFR-BMSCs were treated with ISX-9, and changes in downstream proteins were used to determine whether the ISX-9 worked on the NGFR to transmit signals. The results revealed that the expression of downstream proteins ERK, P-ERK, TAU, and KGF was lower in shNGFR-BMSCs than in BMSCs that had not been treated with ISX-9(Fig. 4e, Supplementary Fig. S3c). The elevated proteins in BMSCs treated with ISX-9 were likewise inhibited in shNGFR-BMSCs at the same time. In short, NGFR may be the first acting target of which ISX-9 induced BMSCS to secrete KGF.

Figure 4.

ISX-9 caused changes in the expression of membrane protein NGFR and downstream proteins in BMSCs. (a) QRT-PCR detects the expression level of NGFR after different concentrations of ISX-9 treated with BMSCs. All data are represented as mean ± SD and the experiment is repeated 3 times independently. The significant difference between the ISX-9 treatment group and the control group was indicated by *P < .05, ***P < 0.001. (b) Western blot verified that ISX-9 acted on BMSCs and upregulated NGFR and its downstream proteins. N = 3. (c) Western blot to verify that the NGFR gene was knocked down in the cells. N = 3. (d) QRT-PCR screening of the sequence with the best infection efficiency. N = 3. Significant differences from controls are indicated by ***P < 0.001. (e) Comparing the effect of ISX-9 on downstream proteins after acting on BMSCs and shNGFR-BMSCs respectively. (f) NGFR gene knockdown efficiency was analyzed by ImageJ.

ISX-9 Regulated KGF Expression in BMSCs Via the NGFR-ERK-TAU-β-Catenin Axis

Numerous investigations have revealed that NGFR activation can result in ERK activation and phosphorylation.²⁰ TAU expression and phosphorylation were regulated by p-ERK.²¹ At the same time, TAU and β-catenin were GSK-3β substrates, and the β-catenin nuclear process regulated KGF transcription106. ERK, an extracellular protein kinase, was essential for signal transmission from the surface receptor to the nucleus, which was typically located in the cytoplasm of cells. There were 2 types of β-catenin: the complex in the cytoplasm and the free body that was about to enter the nuclear.²² Based on the foregoing, we hypothesized that ISX-9 acts on NGFR to regulate the ERK/TAU/β-catenin signaling pathway and targets KGF expression.

To investigate the regulation of KGF expression, ERK and β-catenin inhibitors were used following the ISX-9 action. The results (Fig. 5a, Supplementary Fig. S4a) demonstrated that the expression of KGF was increased by ISX-9. However, this effect was reversed when inhibitors (U0126-ETOH/5 μM) and β-catenin inhibitors (MSAB/5 μM) were used. These findings suggested that ERK and β-catenin were essential in regulating the signal axis that controlled KGF expression. ERK inhibitors (U0126-EtOH/5 μM) had an impact on downstream proteins identified by Western blot. U0126 was shown to reverse the upregulation of ERK, P-ERK, TAU, β-catenin, and KGF, demonstrating that ISX-9 mediated alterations in the Tau/β-catenin/KGF signal axis (Fig. 5b, Supplementary Fig. S4b). The amount of β-catenin in the nucleus rose as a result of the ISX-9 impact. β-catenin inhibitor (MSAB/5 μM) also suppressed the expression of the composite with TCF-4, which targets KGF (Fig. 5c, Supplementary Fig. S4c).

Figure 5.

ISX-9 regulates KGF expression through NGFR-ERK-TAU-β-catenin axis. (a) Both ERK and β-catenin inhibitors significantly reduce KGF expression. Western blot assay of BMSCs treated with ISX-9 (2.5 μM) alone, ISX-9 (2.5 μM) + ERK-inhibitor (U0126/10 μM), ISX-9 (2.5 μM) +β-catenin inhibitor (MSAB/5 μM), ISX-9 (2.5 μM) + ERK-inhibitor (U0126/10 μM) +β-catenin inhibitor (MSAB/5 μM) combination treatment on KGF expression. N = 3. (b) The effect of the ERK inhibitor U0126 (U0126/10 μM) on changes in NGFR downstream proteins brought about by the action of ISX-9 (2.5 μM) was shown by Western blot. N = 3. (c) The effect of β- catenin inhibitor (MSAB/5 μM) on the changes of β-catenin, TCF-4, and KGF in the nucleus induced by ISX-9 (2.5 μM) was shown by Western blot. N = 3. (d) Western blot to confirm the competitive relationship between phosphorylated β-catenin and TAU. N = 3. (e) BMSCs treated with ISX-9 (2.5 μM) by a GSK-3β inhibitor (TSW119/2 μM), results shown by Western blot.

We assumed that TAU and β-catenin were in a competitive interaction in the cytoplasm since they were both GSK-3β substrates and were phosphorylated by GSK-3β. P-TAU rose in response to the rise in TAU expressions. When GSK-3β was limited, phosphorylated β-catenin was reduced, allowing more β-catenin to enter nuclear-targeted KGF transcription. To test this hypothesis, Western blot was used to detect changes in β-catenin and TAU (Fig. 5d, Supplementary Fig. S4d), as well as their phosphorylation. Because of a competitive interaction between β-catenin and TAU as a restricted GSK-3β phosphate as a substrate, p-TAU expression rose while p-β-catenin expression decreased. The GSK-3β inhibitor (TSW119/2 μM) was used to further validate this guess (Fig. 5e, Supplementary Fig. S4e). Following GSK-3β inhibition, the expression of P-TAU and P-β-catenin reduced, but the amount of free β-catenin increased, resulting in enhanced KGF expression. In summary, based on biological data analysis, ISX-9 can considerably boost KGF via the NGFR-ERK-TAU-β-catenin signal axis, as demonstrated by Western Blot and RT-PCR.

BMSCs Work With ISX-9 to Alleviate Acute Lung Injury

A rat ALI model was established to confirm that ISX-9 and BMSCs combined with the repair effect. There were 36 SD rats aged 6-8 weeks, divided into 6 groups at random (Supplementary Fig. S5a). After LPS-induced rats, the wet and wet ratio of lung tissue and the total protein concentration in BALF increased significantly, while SOD content dropped, indicating that dripping LPS in the trachea efficiently developed the ALI model (Supplementary Fig. S5b and S5c). After 7 days of injectable treatment, compared with the PBS group, H&E-stained pulmonary sections indicated that ameliorates LPS-induced acute lung injury alveolar architecture damage and congestion were relieved in BMSCs and in the combination of ISX-9 and BMSCs (Fig. 6a and 6b), along with a decrease in the levels of BALF protein (Fig. 6c). The combination of BMSCs with ISX-9 therapy greatly reduced the production of pro-inflammatory cytokines such as IL-12, TNF-α, IL-1β, and IL-6 (Supplementary Fig. S5d–S5i). ISX-9 combined with shNGFR-BMSCs could lessen the expression of inflammatory factors and the infiltration of inflammatory cells, but there was still a sizable gap in its capacity to repair the alveoli when compared with BMSCs. In short, ISX-9’s assistance with BMSCs to diminish pulmonary edema and reduce inflammatory response was the most significant, which reflected the promotion effect of ISX-9 on transplantation BMSCs treatment ALI. The distribution and role of BMSCS in ALI rats were further observed by live imaging technology (Fig. 6d). Results illustrated that ISX-9 could not only promote the intensity of BMSCs but also delay the rate of BMSCs being cleared by the body (Fig. 6e). Meanwhile, the protein level of KGF in the lung was observed by Western blot and immunofluorescence (Fig. 6f, Supplementary Fig. S5j, S5k). It was discovered that the expression of KGF in the lung after ISX-9 and BMSCs treatment was greatly elevated, which was more remarkable than when treated with ISX-9 and shNGFR-BMSCs combined. ISX-9 may alter BMSCs’s healing effect on lung injury by controlling the expression of NGFR-KGF in the cell, as KGF is a growth factor with mending ability. In conclusion, ISX-9 combined with BMSCs could promote remission of ALI by enhancing the duration and intensity of BMSCs in vivo, while also raising KGF expression to alleviate lung injury.

Figure 6.

ISX-9 combined with BMSCs attenuated LPS-induced lung injury in ALI rats. (a) H&E staining was used to assess the pathological changes in the lung. This image shows that LPS induced pathological changes in lung tissue and that administration of BMSCs and ISX-9 attenuated lung injury (×100/×400 magnification). (b) Quantitative analysis of lung injury scores for each group. All data were repeated 3 times. (c) Protein concentration in alveolar lavage fluid in different treatment groups. N = 3. (d) PE IVIS Spectrum CT small animal live imager to observe the distribution and intensity of the action of BMSCs in vivo, the luminescence site is BMSCs, the darker red color represents the stronger action intensity. N = 4. (e) Living image software analysis of the mean fluorescence intensity. On DAY6, the ISX-9 combined with BMSCs treatment group compared with BMSCs alone group, **P < .01. (f) Immunofluorescence of tissue sections showing KGF expression in lung tissue. #: normal group vs LPS-induced ALI group; *LPS-induced ALI group vs treatment group. Relative mRNA levels in the normal group of rats were normalized by the 2-ΔΔCt method. Compared with normal control, #P < .05, ##P < .01 ###P < .001;compared with the ALI model group,*P < .05,**P < .01,***P < .001; compared with the ISX-9 + BMSCs group, &P < .05, &&P < .01, &&&P < .001. The data results were repeated 3 times.

Discussion

Mesenchymal stem cells (MSCs) have been extensively researched for their potential to treat acute lung injury (ALI) due to their strong paracrine function.²³ Studies have shown that MSCs secreted KGF to accelerate the regeneration of epithelial cells and reverse pulmonary fibrosis.²⁴ However, the paracrine function of MSCs was diminished in the inflammatory microenvironment.²⁵ In our previous study, MSCs combined with liraglutide were observed to expedite the repair of ALI by increasing the paracrine secretion of cytokines such as KGF,¹⁴ Ang-1, and SFTPC(surfactant protein C).²⁶ The goal of this study was to identify compounds that promote paracrine KGF from MSCs via compound screening.

A cell model targeting the KGF promoter was created and the luciferase activity was used to determine the extent of KGF promoter activation. Human-derived cells were used in compound screening to provide more reliable evidence for MSCs allograft clinical applications in the future.²⁷ Previous studies have used screening compound models developed from MSCs to screen compounds that aid in regeneration,²⁸ reduce liver damage, or treat neurological diseases.²⁹ However, our study presented a novel approach by proposing MSCs as a cell model for screening compounds that promote the paracrine function of MSCs. This approach aimed to enhance the effectiveness of MSCs in treating ALI, making it a pioneering study in this field.

In the compound library, we looked at 9 compounds that activate the KGF promoter. These are ISX-9, cardiogenol C, CH 223191, PD 98059, GW 788388, ID 8, kenpaullone, BIO-acetoxime, and valproic acid. In this study, it was found that compound ISX-9 significantly increased the activity of luciferase by 61.9 times compared to the control group. Additionally, the degree of ISX-9 activating KGF expression was the most significant. Through the verification of 9 candidate compounds at the mRNA and protein levels, it was concluded that ISX-9 was the most effective compound in promoting the secretion of KGF from MSCs through comprehensive screening. ISX-9 is a neurogenic chemical that has been shown to convert neural stem cells into neurons. However, there have been no studies linking ISX-9 to the paracrine function of MSCs nor has the effect of ISX-9 on LPS been studied. Based on screening results that showed ISX-9 promoting the expression of KGF in MSCs, we used rat-derived BMSCs as the main research cells to facilitate subsequent exploration of its mechanism of action.³⁰

Quantitative proteomic analysis of TMT-labeled differential proteins of BMSCs treated with ISX-9 was used to investigate its effect on the paracrine ability of BMSCs to produce KGF. Our findings suggest that ISX-9 regulates the NGFR/ERK/TAU/β-catenin axis, ultimately leading to the promotion of KGF production. Notably, we observed that upregulated proteins in the differential proteins were enriched in the biological functions of neurotrophin signaling pathways and microtubule binding. Among the significantly upregulated proteins, NGFR and Mapt were identified as potential targets for further investigation.

The NGFR-ERK-TAU-β-catenin signaling axis was proposed based on the analysis of differential protein results. The NGFR gene had been knocked out and ISX-9 had been applied to normal BMSCs and shNGFR-BMSCs. It revealed that ISX-9 induced a series of protein expression increases in BMSCs that were reversed after NGFR knockdown, thus validating the proposed assumption. Furthermore, previous studies have shown that NGFR antibody blockers inhibited the phosphorylation of TAU, which suggested that Aβ-induced TAU is regulated by NGFR signaling.³¹ It has also been proposed that phosphorylation of Tau resulted in decreased phosphorylation of β-catenin and increased nuclear translocation of β-catenin.³² This suggested competitive phosphorylation of β-catenin by GSK-3β, which promotes β-catenin function. To verify the downstream protein interactions in this signaling axis, we used signaling pathway inhibitors. Our findings indicated that ERK and its p-ERK can affect the changes of the downstream protein TAU when ERK inhibitors are used. The direct relationship between β-catenin and KGF expression was confirmed through the use of a β-catenin inhibitor, while our hypothesis was supported by the use of a GSK-3β inhibitor. Following the administration of the GSK-3β inhibitor, there was a decrease in p-β-catenin and an increase in free β-catenin, leading to the promotion of β-catenin into the nucleus and a significant increase in KGF expression. These experimental results demonstrated that ISX-9 targets BMSCs to secrete KGF by regulating the NGFR-ERK-TAU-β-catenin signaling axis.

Ultimately, an ALI model was established to evaluate the therapeutic potential of ISX-9 in combination with BMSCs. Our findings revealed a synergistic effect of ISX-9 with BMSCs for ALI treatment, as evidenced by the reduction in inflammatory factors post-treatment. The reduction of BMSCs inflammatory response by ISX-9 was found to be due to increased expression of KGF. Further comparison between ISX-9 combined with BMSCs and shNGFR-BMSCs confirmed that ISX-9 regulated KGF expression by acting on NGFR, thereby aiding in the repair of ALI. Most notably, this study demonstrated that ISX-9 combined with BMSCs promotes BMSCs’ homing properties by promoting the duration and intensity of BMSCs in the lung.

While this study demonstrated the potential of ISX-9 to enhance the therapeutic effects of BMSCs in ALI by promoting the secretion of KGF, it is important to note that the study does not investigate the direct binding of ISX-9 to BMSCs or the specific interaction site. Therefore, further research was needed to understand the mechanism of ISX-9 interaction with BMSCs in vivo.

Conclusion

In this study, we identified the ISX-9 as a potential enhancer of MSCs’ ability to secrete KGF, thereby promoting their application in repairing acute lung injury. Our findings demonstrated that ISX-9 could regulate KGF secretion from MSCs through the NGFR-ERK-TAU-β-catenin signaling axis in vitro and reduce inflammation in the ALI model.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82170086), Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases (20dz2261100), National Key R&D Program of China(2022YFA1304300), Construction of a cohort database and biological sample holographic database for severe pneumonia (SHDC2020CR5010).

Conflict of Interest

The authors declared no potential conflicts of interest.

Author Contributions

Y.T.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing. Q.D.: collection and assembly of data. X.Y.: Provision of study material. C.W.: administrative support. V.M.L.: manuscript writing. R.J.: conception and design. X.L.: conception and design, financial support, manuscript writing. Y.F.: conception and design, financial support, manuscript writing, final approval of manuscript data.

Data Availability

The data underlying this article are available in the article and in its online supplementary material.