Cell culture and treatment

The human primary type II alveolar epithelial cell line (AT II) was obtained from Procell Co. Ltd (CP-H209, China). F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution was applied to maintain cell growth. Cells were placed in a humidified incubator at 37oC with 5% CO₂, and the culture medium was refreshed every 2 to 3 days.

To establish ALI in vitro, AT II cells were treated with LPS (10 µg/ml, HY-D1056, MedChemExpress, USA) for 24 h and then replaced with a normal medium [27]. SRPIN340 (an SRPK1 inhibitor) was used to determine the role of SRPK1 in ALI, by which AT II cells were pretreated with SRPIN340 (5, 10, 15, and 20 µM, HY-13949, MedChemExpress, USA) for 24 h before subsequent experiments were performed.

Cell transfection

The short hairpin RNAs (shRNAs) against KLF7 and LIMK1 expression were constructed by GenePharma Co. Ltd (China), as well as the overexpression plasmid of KLF7 and LIMK1. Specifically, the AT II cells were pre-seeded in a 6-well plate, and the transfection process started once the cell confluence was around 60-70%. All the plasmids packaged with lentivirus were transfected into AT II cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, USA) according to the kit instructions [28]. In brief, Lipofectamine 3000 was diluted with Opti-MEM medium. Subsequently, the plasmid DNA was mixed with an appropriate quantity of Opti-MEM medium and P3000 reagent. Following the combination of the two reagent solutions, the resulting mixture was administered to the cells. After 24 h of transfection, 3 µg/ml of puromycin (P8833, Sigma Aldrich, USA) was used to select cells, and then the transfection efficiency was tested for subsequent experiments.

Real-time quantitative reverse transcription PCR (RT-qPCR)

Total RNA was extracted from cell samples using an RNA isolator (R401-01, Vazyme, China). The HiScript II 1st Strand cDNA Synthesis Kit (R212-01, Vazyme, China) was employed to reverse transcribe the extracted RNA into complementary DNA (cDNA). Following this, RNase-free ddH₂O was used to prepare a 20 µl reaction mixture for real-time PCR (Roche LightCycler 96, Switzerland), which included the cDNA template, primers, and 2×SuperReal PreMix Plus (FP205-01, TIANGEN, China). β-actin was designated as the internal reference gene to compare gene expression variations. The primer sequences used in this study were as follows (5´-3´):

Western blot

To obtain the total protein of cell samples, RIPA lysis buffer (P0013B, Beyotime, China) mixed with protease and phosphatase inhibitors was used. After quantifying the protein concentration using a BCA analysis kit (P0010S, Beyotime, China), an appropriate volume of sodium dodecyl sulfate (SDS) loading buffer (P0015L, Beyotime, China) was added to the protein sample. Subsequently, an SDS-PAGE gel was prepared for electrophoretic separation. After the separation process, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (88518, Thermo Scientific, USA). The membrane was then soaked and blocked in 5% skim milk for 1 h at room temperature. Following this, the membrane was respectively exposed to the primary antibodies against KLF7 (sc-398576, Santa Cruz Biotechnology, USA), LIMK1 (ab95186, Abcam, UK), SRPK1 (ab90527, Abcam, UK), p-SRPK1 (PA5-106156, Invitrogen, USA), and β-actin (ab8227, Abcam, UK) for overnight incubation at 4oC. After the removal of the primary antibody, the membrane was treated with the horse radish peroxidase (HRP)-conjugated secondary antibodies (ab288151, ab6728, Abcam, UK). Finally, the proteins were visualized with the help of ECL chromogenic solution under an imaging system (Azure 600, USA), and the results were analyzed using ImageJ (version: 1.8.0, National Institutes of Health, USA).

Enzyme-linked immunosorbent assay (ELISA)

In brief, the collected cell supernatant and biotin- labeled antibody were incubated simultaneously. After washing, avidin-labeled HRP was added. Following another incubation and washing phase, substrates A and B were introduced with the concurrent action of the enzyme conjugate. Finally, the sample to be tested was placed into a microplate reader (Thermo Fisher Scientific, USA) to detect absorbance at 450 nm. The ELISA kits of TNF-α (ml077385), IL-1β (ml058059), and IL-6 (ml058097) were purchased from Enzyme-linked Biotechnology Co., Ltd (China).

CCK-8 assay

The Cell Counting Kit-8 (CCK-8) assay was employed to assess alterations in the viability of AT II cells after treatment with LPS and/or transfection with related plasmids. Briefly, AT II cells were seeded into a 96-well plate, with an approximate density of 2 × 10⁴ cells per well. 24 hours later, 10 µl of CCK-8 reagent (C0037, Beyotime, China) was added to each well for assessment. The wells were then incubated at 37oC for an additional hour, after which the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

Flow cytometry for cell apoptosis detection

Cells were harvested using ethylenediaminetetraacetic acid (EDTA)-free trypsin digestion. Following a washing step, the cells were resuspended in a binding solution, to which Annexin V-FITC (C1062S, Beyotime, China) and propidium iodide (C1062S, Beyotime, China) were added and thoroughly mixed. The mixture was incubated at room temperature without light for at least 20 min. Subsequently, flow cytometry (Beckman Coulter, USA) was used to assess cell apoptosis within one hour, and the data were analyzed using FlowJo software (version: 10.8.1, Tree Star, USA).

Dual-luciferase reporter assay

The wild-type and mutant LIMK1 promoter fragments were inserted into the pGL3-basic vector (Promega, USA) to generate various dual-luciferase reporter plasmids. Subsequently, the constructed plasmid vector was co-transfected into AT II cells alongside either the sh-NC or sh-KLF7 plasmid. Twenty-four hours after transfection, firefly and Renilla luciferase activities were measured using the Dual- Glo system (E2920, Promega, USA), and the ratio of luciferase activities between the two groups was calculated to assess transcriptional activity.

Chromatin immunoprecipitation (ChIP)

This assay applied the Pierce Magnetic ChIP Kit (26157, Thermo Fisher Scientific, USA) [29]. In summary, cells were treated with a culture medium containing a 1% formaldehyde solution at 37oC for 10 minutes, after which the cross-linking reaction was halted using glycine. Subsequently, the cells were lysed and the chromatin was fragmented using a sonicator. Following this, the nuclear pellet was resuspended in an IP dilution buffer, and either IgG (ab172730, Abcam, UK) or KLF7 antibody (sc-398576, Santa Cruz Biotechnology, USA) was introduced and incubated overnight at 4oC. After incubation, 20 µl of magnetic beads were added to each IP sample and incubated for an additional 2 hours at 4oC with gentle mixing. The washed beads were then eluted using IP elution buffer to isolate the DNA-protein complexes. Finally, purified DNA was obtained through a DNA Recovery process, allowing for subsequent qPCR analysis.

Glutathione-S-transferase (GST) pull-down assay

The plasmid with the GST tag and SRPK1 sequence was introduced into BL21 competent cells, which were cultured overnight. The bacterial culture from the positive clone was then expanded. Following the collection of the bacterial cells, they were resuspended in a buffer supplemented with protease inhibitors, and bacterial lysis was achieved through sonication. The supernatant was collected after centrifugation. The prepared cell lysate was then combined with the GST fusion bait protein and incubated at 4oC with gentle agitation overnight. The following day, glutathione-agarose beads were added to the mixture, which was shaken and incubated for 4 hours at 4oC. The beads were washed repeatedly, and the interacting protein complex was subsequently eluted from the beads using an elution buffer. Verification and identification of the eluted proteins were conducted using Western blot analysis.

Co-immunoprecipitation (Co-IP)

This experiment was carried out using the Pierce Classic Magnetic IP/Co-IP Kit (88804, Thermo Fisher Scientific, USA) [30]. In brief, AT II cells were treated with RIPA lysis buffer at a low temperature for 30 min to isolate the protein. Subsequently, the cell lysates were incubated overnight at 4oC with IgG (ab172730, Abcam, UK) or the antibody against LIMK1 (ab95186, Abcam, UK) to ensure complete binding. The following day, the protein-antibody mixture was added to a centrifuge tube containing Protein A/G beads and incubated at room temperature for 2 hours. The beads were then collected using a magnetic stand, and unbound samples were removed. The magnetic beads were washed multiple times using IP lysis/wash buffer, and the precipitate was eluted using an elution buffer. The obtained samples were analyzed through immunoblotting experiments to assess protein-protein interactions.

Statistical analysis

All data are presented as mean ± standard deviation, and the analytical procedures were conducted using GraphPad Prism 9.0 software (version: 9.0, GraphPad Software, USA). Student’s t-test was employed to assess differences in continuous variables between two groups. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s test was used. Each experiment was conducted three times to ensure reliability. A p-value of less than 0.05 was deemed statistically significant.

Knockdown of KLF7 inhibited LPS-induced inflammatory response and apoptosis of AT II cells

Our initial study found that the mRNA and protein levels of KLF7 were elevated in AT II cells treated with LPS (Fig. 1A, B). To confirm the role of KLF7, we further constructed a KLF7 knockdown vector and infected it into AT II cells. The results, shown in Figure 1C, D, illustrated the efficiency of KLF7 knockdown. On this basis, we further induced ALI in cells. As displayed in Figure 1E, F, KLF7 inhibition significantly counteracted the increased KLF7 expression induced by LPS treatment in AT II cells. LPS treatment caused an increase in the expression and release levels of the inflammatory mediators TNF-α, IL-1β, and IL-6, and their levels decreased when KLF7 declined (Fig. 1G, H). Furthermore, the viability of AT II cells was suppressed by LPS treatment, and the reduction of KLF7 attenuated the observed decrease in cell viability (Fig. 1I). Finally, as shown in Figure 1J, KLF7 suppression also reversed the effect of elevated cell apoptosis induced by LPS treatment. These results demonstrated that KLF7 has a pro-inflammatory function in LPS-induced AT II cells.

Fig. 1

Knockdown of KLF7 inhibited LPS-induced inflammatory response and apoptosis of AT II cells. A, B) AT II cells were treated with LPS (10 μg/ml) for 24 h to induce ALI conditions. RT-qPCR and Western blot were applied to detect KLF7 expression. C, D) sh-KLF7 and its control sh-NC were respectively transfected into AT II cells. RT-qPCR and Western blot analysis were conducted to check the efficiency of KLF7 knockdown. After the transfection of sh-KLF7 and sh-NC, AT II cells were treated with LPS. E, F) RT-qPCR and Western blot were used to measure KLF7 levels. G) RT-qPCR was used to measure the expression levels of TNF-α, IL-1β, and IL-6. H) The secretion levels of TNF-α, IL-1β, and IL-6 were tested using their own ELISA kits. I) CCK-8 assay was applied to determine the viability of AT II cells. J) Flow cytometry was used to quantify cell apoptosis levels. All experiments were conducted in triplicate. **p < 0.01, ***p < 0.001

KLF7 promoted LIMK1 transcriptional activation in AT II cells

As illustrated in Figure 2A, B, both mRNA and protein expressions of LIMK1 were increased under ALI conditions. Subsequently, we investigated the potential regulatory interaction between KLF7 and LIMK1. The results, shown in Figure 2C, display the binding sites between the KLF7 and LIMK1 promoter, which were predicted using the JASPAR database. Dual-luciferase assay showed that KLF7 knockdown resulted in a marked reduction in the transcriptional activity of LIMK1, which was abolished when LIMK1 was mutated (Fig. 2D). In addition, KLF7 was greatly enriched in the promoter region of LIMK1 (Fig. 2E). The above results indicated that KLF7 and LIMK1 promoters have a mutual binding relationship. Subsequently, we investigated the effect of KLF7 interference on LIMK1 levels. As illustrated in Figure 2F, G, oe-KLF7 significantly increased KLF7 mRNA and protein levels. KLF7 overexpression increased the mRNA and protein levels of LIMK1 (Fig. 2H, I). In contrast, knocking down KLF7 significantly reduced LIMK1 levels (Fig. 2J, K). As depicted in Figure 2L, M, LIMK1 was found to be upregulated following treatment with LPS, whereas the knockdown of KLF7 abolished this increase. The above findings suggested that KLF7 can bind to the LIMK1 promoter region and then enhance LIMK1 transcription and translation in AT II cells.

Fig. 2

KLF7 promoted LIMK1 transcriptional activation in AT II cells. A, B) AT II cells were treated with LPS (10 μg/ml) for 24 h to induce ALI conditions. RT-qPCR and Western blot were applied to evaluate LIMK1 expression. C) JASPAR database predicted the potential binding site of KLF7 and the LIMK1 promoter. D) Dual-luciferase reporter assay was conducted to test the transcriptional activity of LIMK1 after KLF7 knockdown. E) ChIP experiment was used to confirm the interaction between KLF7 and the LIMK1 promoter. oe-KLF7 and its corresponding control plasmids were respectively transfected into AT II cells. F, G) KLF7 overexpression effect was analyzed by RT-qPCR and Western blot. H, I) LIMK1 expression was assessed by RT-qPCR and Western blot. J, K) LIMK1 expression was assessed by RT-qPCR and Western blot after AT II cells were transfected with sh-KLF7 and its corresponding control plasmids. L, M) AT II cells were treated with LPS after transfection of sh-KLF7. LIMK1 expression was quantified using RT-qPCR and Western blot. All experiments were conducted in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

LIMK1 overexpression reversed the effect of KLF7 silencing on LPS-induced inflammatory response and apoptosis of AT II cells

Subsequently, by overexpressing LIMK1 in AT II cells, we explored the role of the KLF7/LIMK1 pathway in ALI. As shown in Figure 3A, B, the transfection of LIMK1 overexpression plasmids resulted in a substantial increase in LIMK1 expression within AT II cells. The increase in LIMK1 levels induced by LPS was eliminated by KLF7 downregulation, and overexpression of LIMK1 further reversed the impact of KLF7 knockdown on LIMK1 expression (Fig. 3C, D). Similarly, LIMK1 overexpression also affected the inflammatory response in AT II cells after LPS treatment. The results, shown in Figure 3E, F, revealed that LIMK1 upregulation elevated the reduced TNF-α, IL-1β, and IL-6 levels caused by KLF7 downregulation. Moreover, the enhanced viability of AT II cells associated with KLF7 deficiency was subsequently diminished by LIMK1 elevation (Fig. 3G). Concurrently, LIMK1 upregulation was found to abolish the influence of KLF7 knockdown on the apoptosis of AT II cells, that is, to exacerbate the apoptosis in these cells (Fig. 3H). This evidence indicated that KLF7 upregulates LIMK1 to facilitate the inflammatory response and apoptosis of AT II cells induced by LPS.

Fig. 3

LIMK1 overexpression reversed the effect of KLF7 silencing on LPS-induced inflammatory response and apoptosis of AT II cells. A, B) LIMK1 was overexpressed in AT II cells. LIMK1 overexpression efficiency was verified by RT-qPCR and Western Blot. KLF7 was knocked down or/and LIMK1 was overexpressed in ALI cell models. C, D) RT-qPCR and Western Blot were applied to assess LIMK1 expression. E) RT-qPCR was utilized to determine TNF-α, IL-1β and IL-6 expressions. F) ELISA assays were applied to measure TNF-α, IL-1β and IL-6 secretion. G) CCK-8 assay was conducted to evaluate cell viability. H) Detection of cell apoptosis was performed by flow cytometry. All experiments were conducted in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

LIMK1 promoted phosphorylation of SRPK1

We continued to investigate the downstream molecules associated with LIMK1 in the context of ALI. Using the HitPredict and BioGRID databases, we predicted a potential interaction between the LIMK1 and SRPK1 proteins. SRPK1 has been proven to act as a pro-inflammatory factor in ALI [25]. LIMK1 is known to regulate protein phosphorylation levels as a phosphokinase [31]. Thus, we further detected changes in SRPK1 protein phosphorylation levels under LPS treatment. As shown in Figure 4A, an increase in both the protein levels and phosphorylation of SRPK1 in LPS-treated AT II cells was observed. Next, we validated the interaction between LIMK1 and SRPK1 through GST pull-down and Co-IP assays (Fig. 4B, C). Following this, we performed a knockdown of LIMK1 in AT II cells. As illustrated in Figure 4D, E, LIMK1 silencing caused a significant reduction in LIMK1 expression. Western blot analysis revealed that the knockdown of LIMK1 specifically affected the phosphorylation level of SRPK1 without altering its overall protein expression (Fig. 4E). Further experiments involving LPS treatment demonstrated that LPS enhanced the phosphorylation activity of SRPK1, and LIMK1 inhibition mitigated the increase in SRPK1 phosphorylation induced by LPS (Fig. 4F). Collectively, LIMK1 interacts with SRPK1 protein in AT II cells to promote its phosphorylation.

Fig. 4

LIMK1 promoted the phosphorylation of SRPK1. A) AT II cells were treated with LPS. Western blot determined the protein and phosphorylation levels of SRPK1. B, C) GST pull-down and Co-IP assays were applied to validate the protein binding relationship between LIMK1 and SRPK1. LIMK1 was silenced in AT II cells. D) RT-qPCR was employed to assess LIMK1 knockdown efficiency. E) Western blot analysis for LIMK1, SRPK1 expression and SRPK1 phosphorylation level. F) LIMK1 was silenced in LPS-treated AT II cells. Western blot was performed to quantify LIMK1, SRPK1 and SRPK1 phosphorylation levels. All experiments were conducted in triplicate. **p < 0.01, ***p < 0.001

KLF7 stimulated the LPS-induced inflammatory response and cell apoptosis by regulating the LIMK1/SRPK1 axis

Finally, we identified the regulatory function of the KLF7/LIMK1/SRPK1 pathway in ALI. First, the influence of SRPIN340 (SRPK1 inhibitor) on cellular viability was assessed. We observed that 5 µM and 10 µM of SRPIN340 did not produce a significant effect on cell viability. When the concentration reached 15 µM, it reduced cell viability (Suppl. Fig. 1). Since 10 µM SRPIN340 has no cytotoxic effect, this concentration was used for further exploration. As shown in Figure 5A, SRPIN340 reduced SRPK1 phosphorylation in LPS treated AT II cells; LIMK1 upregulation counteracted the inhibitory impact of KLF7 knockdown on SRPK1 activity, while SRPIN340 abolished the effects of LIMK1 overexpression. Furthermore, KLF7 knockdown and SRPIN340 treatment resulted in a reduction in the expression and secretion of inflammatory mediators, respectively. Conversely, the overexpression of LIMK1 antagonized the impact of KLF7 knockdown, while SRPIN340 further mitigated the effects induced by LIMK1 overexpression (Fig. 5B, C). In addition, overexpression of LIMK1 reversed the enhancement effect of KLF7 knockdown on cell viability, while inhibition of SRPK1 further antagonized the decreased effect of overexpression of LIMK1 on cell viability (Fig. 5D). Then flow cytometry was employed to assess cell apoptosis. As illustrated in Figure 5E, SRPIN340 suppressed the cell apoptosis induced by LPS; moreover, LIMK1 upregulation was observed to counteract the anti-apoptotic effects resulting from KLF7 knockdown, and SRPIN340 abolished the impact of LIMK1 overexpression again. In conclusion, KLF7 aggravated the inflammatory response and apoptosis in LPS-treated AT II cells via the LIMK1/SRPK1 pathway.

Fig. 5

KLF7 stimulated LPS-induced inflammatory response and cell apoptosis by regulating the LIMK1/SRPK1 axis. AT II cells were treated with sh-KLF7, oe-LIMK1, and/or SRPIN340, followed by LPS treatment. A) Western blot was applied to determine the phosphorylation of SRPK1. B) RT-qPCR was used to detect the expression TNF-α, IL-1β, and IL-6. C) ELISA assays were applied to assess the secretion of TNF-α, IL-1β, and IL-6. D) Cell viability was evaluated by CCK-8 assay. E) Cell apoptosis was measured by flow cytometry. All experiments were conducted in triplicate. **p < 0.01, ***p < 0.001