Inhibition of STAT3 alleviates LPS-induced apoptosis and inflammation in renal tubular epithelial cells by transcriptionally down-regulating TASL

The gene expression microarrays of individuals with SLE and normal healthy individuals were retrieved and then normalized by resequencing microarray (RMA). The differentially expressed genes (DEGs) were screened using the online tool GEO2R. Using |log₂fold change (FC)|≥ 1 and adj. P < 0.05 as screening criteria, DEGs were obtained between patients with SLE and normal healthy individuals. The volcano plot was obtained using the R language package ggplot2. Twenty DEGs were selected, and the heatmap was plotted using GraphPad 9.0. To conduct a more detailed analysis of the identified DEGs, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed using the tool Metascape (https://​metascape.​org/​). Protein–protein interaction (PPI) network studies were generated for DEGs utilizing STRING (https://​cn.​string-db.​org).

Peripheral blood of 6 LN patients and 5 healthy controls were collected. Ethylenediaminetetraacetic acid (EDTA) anticoagulation tubes were used to collect 2 mL whole blood samples from pediatric subjects with SLE and healthy pediatric subjects. The mononuclear cells were immediately extracted for RNA and detected for TASL and STAT3 expression. The approval for study protocols was granted by the Institutional Review Board of the Wuxi Children's Hospital Affiliated to Jiangnan University (WXCH2021-12-004). In addition, all the legal guardians of included children signed written informed consents before sample collection.

Human renal tubular epithelial cell line human kidney 2 (HK2) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in an incubator at 37 °C with 5% CO₂. The cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% antibiotic (penicillin/streptomycin). To simulate inflammation during LN model establishment, we treated HK2 cells with lipopolysaccharide (LPS) using the method described in the literature of Xu et al., to evaluate the effect produced by TASL during inflammation [23]. The same volume of phosphate-buffered saline (PBS) solution was used as a control.

Plasmid transfection was performed according to the instructions provided by Lipofectamine™ 3000 (Thermo Fisher). HEK293T and HK2 cells were seeded in well plates and cultured using penicillin and streptomycin-free medium. Transfection was performed when the cells grew to approximately 70–80% confluence. RNA samples were extracted after 24 h, and protein samples were extracted after 48 h.

A dual-luciferase reporter gene assay kit (Promega, USA) was employed for the detection of luciferase activity. The STAT3 overexpression plasmid and small interfering RNA (siRNA) targeting STAT3 (siSTAT3) were co-transfected with the recombinant plasmid (Jinbeijin Biotechnology Co., Ltd., China) containing the TASL promoter fragment of luciferase and the internal reference plasmid (pRL-TK, Promega, USA), respectively. Luciferase activity was then assayed as per the provided guidelines. The firefly luciferase activity was normalized to Renilla luciferase activity.

The RNA-Quick Purification Kit (YISHAN Biotechnology., LTD, China) was utilized to extract total RNA, and PrimeScript RT Master Mix (TaKaRa, Japan) was employed for the synthesis of cDNA. The SYBR Green RT-PCR kit (Vazyme, China) was used to conduct fluorescence qRT-PCR assays. Relative gene expression was identified using the 2^−ΔΔCt method and normalized to GAPDH. Primers for TASL, STAT3, BCL-2, BAX, TNF-α, and IL6 were designed by TSINGKE (Beijing, China).

The ChIP kit (Millipore, USA) was used for this assay, and DNA was purified and precipitated as per the provided guidelines for subsequent PCR analysis. PCR products were detected by electrophoresis on agarose gels. The SYBR kit (Vazyme, China) was employed to perform quantitative PCR of ChIP DNA.

The cell viability was determined by means of the cell counting kit-8 (CCK-8) (MCE, USA) as per the provided guidelines. Optical density (OD) values at 450 nm were quantified using a microplate reader (Thermo Fisher, USA), followed by a viability analysis.

The Annexin V-FITC/ PI staining kit (Vazyme, China) was used, and the tested cells were processed per the provided guidelines. In addition, a flow cytometer (Beckman Coulter, USA) was utilized in order to conduct an apoptosis assay.

Using a fixation solution of 4% paraformaldehyde, the cells in the well plates were fixed for a duration of 30 min, following a 5-min treatment with 0.3% Triton X-100 at room temperature. Next, cells were subjected to incubation with TUNEL reagent (Beyotime, China) for 60 min. Finally, fluorescence microscopy was employed for the observation of cells sealed with a sealing agent containing DAPI.

Levels of inflammatory factors IL6 and TNF-α in cell supernatants were quantified with ELISA kits and then detected using a fully automated biochemical analyzer ADVIA® 2400 (Siemens, China).

After lysis buffer-based extraction of total proteins, equal amounts of protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Afterward, 5% bovine serum albumin (BSA) was used as a blocking agent for 1 h at room temperature. The blots were probed with primary antibodies overnight at 4 °C. A horseradish peroxidase-conjugated secondary antibody (diluted at 1:5000) was subsequently introduced. Enhanced chemiluminescence reagents (Thermo Fisher Scientific, USA) were employed to visualize the protein bands. Primary antibodies, including anti-STAT3, anti-TASL, anti-cleaved caspase-3, anti-BAX, anti-Cleaved-Caspase3 and anti-BCL-2 were purchased from Abcam. Anti-β-actin antibody (loading control) and goat anti-rabbit IgG H&L (HRP) (secondary antibody) were purchased from Beyotime.

The experimental results were presented as mean ± standard deviation, and GraphPad prism 9.0 software was employed for statistical analysis. The data between two groups were compared using an unpaired t-test, and P < 0.05 was considered statistically significantly different.

In this study, the study subjects of the GSE185047 dataset were retrieved, and the microarray sequencing results were normalized. A total of 2620 DEGs, including 1301 down-regulated genes and 1319 up-regulated genes, were analyzed using the GEO2R tool. Among them, 20 DEGs were found to be significantly differentially expressed in peripheral blood mononuclear cells (PBMCs) of adult individuals with SLE compared with healthy individuals (Fig. 1A). The GO and KEGG analyses highlighted that DEGs were mainly enriched in such pathways as “intrinsic immune response, cytokines, immune response regulatory signaling pathway, leukocyte activation regulation, stress response regulation, kinase binding protein regulation” and other pathways (Fig. 1B). To further search for key nodes of DEGs, the genes were uploaded to the STRING online database to construct the PPI network. The results showed that the PPI network contained 32 node molecules, and TLR4, TLR8, and IFI16 were the core nodes of the PPI network, including the TLR8 molecular adaptor protein CXorf21 (Fig. 1C). The function of CXorf21 in SLE pathogenesis has been under investigation for several years, but the role of its protein product remained unclear until Heinz et al. discovered it to be an adaptor protein in TLR signaling contributing to the progression of SLE in 2020 [15]. Then, the gene was given a new name, TASL [15]. By analyzing the GSE185047 dataset, it was found that the expression of STAT3 and TASL was notably elevated in individuals with SLE than in healthy controls (Fig. 1D). STAT3 expression is critically involved in SLE and is associated with multi-organ damage [24]. Additional analysis indicated a strong positive relationship between the expression of STAT3 and TASL (Fig. 1E). Subsequently, the detection of RNA expression in PBMCs in healthy and diseased pediatric subjects highlighted that the mRNA expression of TASL and STAT3 was considerably elevated in children with SLE than in healthy children (Fig. 1F). The Pearson correlation analysis revealed a positive relationship between the expression of STAT3 and TASL in children with SLE (Fig. 1G). These results highlight that TASL triggers the development of SLE, and its gene expression is correlated with STAT3.

The application of the JASPAR website predicted the presence of two STAT3 binding sites in the region from − 940 bp to + 60 bp of the TASL promoter (Fig. 2A and Additional file 1: Fig. S1A). The TASL gene promoter from − 940 bp to + 60 bp was cloned into the pGL3-Basic luciferase reporter gene plasmid (Additional file 1: Fig. S1B), and the product of the recombinant plasmid digested by double digestion with KpnI and NheI restriction endonucleases was subsequently subjected to agarose gel electrophoresis. The results showed that two specific bands appeared at around 4.8 kb and 1 kb, indicating that the TASL promoter fragment-containing pGL3-Basic plasmid was successfully constructed (Additional file 1: Fig. S1C). The constructed pGL3-Basic vector containing the TASL promoter fragment and the negative control (NC) pGL3-Basic plasmid were transfected into HEK293T and HK2 cells. The luciferase reporter gene assay showed that the luciferase activity of the gene with the TASL promoter fragment was considerably higher compared with the NC pGL3-Basic (Additional file 1: Fig. S1D), indicating that the region from − 940 bp to + 60 bp of the TASL gene is a functional promoter. The TASL promoter-containing gene plasmid, along with siSTAT3, control siRNA, pSTAT3 or pENTER were transfected into HEK293T cells and HK2 cells, respectively. The results indicated that the TASL gene promoter activity was markedly reduced in the siSTAT3 group when compared with the control siRNA, while the overexpression of STAT3 in the pSTAT3 group increased the human TASL gene promoter activity (Fig. 2B). Subsequently, point mutant plasmids were constructed separately for the two binding sites of the human TASL gene with STAT3, named STAT3-mutant (mut)A and STAT3-mutB. Additionally, simultaneous mutations were introduced to STAT3-mutA and STAT3-mutB, resulting in a mutant named STAT3-mutC. Compared with the original plasmid, promoter activities were decreased by 48.6% after the mutation of STAT3-mutA alone and by 45.5% after the mutation of STAT3-mutB alone. It was decreased by 51.7% when both sites were mutated simultaneously, with no further significant decrease compared to single-point mutation (Fig. 2C). To further clarify the binding of transcription factor STAT3 to TASL promoter, ChIP-qPCR and agarose gel electrophoresis in HK2 cells indicated that the binding of STAT3 to TASL promoter region was notably elevated than that of non-specific IgG control (Fig. 2D). To investigate the impact of STAT3 on TASL mRNA and protein expression, siSTAT3 and pSTAT3 were separately transfected into HK2 cells. qRT-PCR and Western blot findings highlighted that TASL mRNA and protein levels were considerably decreased in the siSTAT3 group compared with the NC group, while they were remarkably elevated in the pSTAT3 group (Fig. 2E–H).

Multiple studies have adopted LPS-induced HK2 cells to create an in vitro LN cell model [23]. Cell viability was measured after LPS stimulation of HK2 cells, and the results showed that cell viability was elevated after 10 μg/mL LPS treatment for 24 h (Additional file 1: Fig. S1E). Western blot assay highlighted that LPS induced a considerable decrease in BCL-2 levels and increased protein levels of cleaved caspase-3, BAX, STAT3, and TASL in HK2 cells (Fig. 3A). Meanwhile, qRT-PCR assay revealed that mRNA expression of BAX, STAT3, and TASL was elevated, and that of BCL-2 was decreased (Fig. 3B). In addition, ELISA highlighted that the inflammatory factors TNF-α and IL6 levels were notably enhanced in the supernatant of LPS-treated HK2 cells (Fig. 3C), while the mRNA expression of TNF-α and IL6 was also enhanced simultaneously (Fig. 3D). TUNEL-based analysis indicated that LPS treatment resulted in a considerable increase in apoptotic cells (Fig. 3E). In conclusion, the expression of STAT3 and TASL was elevated, and cell apoptosis and inflammation were also increased in the LPS-induced in vitro LN model.

After transfection of siTASL into HK2 cells, mRNA and protein levels of TASL were considerably reduced (Fig. 4A, B). Silencing of TASL in LPS-induced HK2 cells significantly down-regulated protein levels of cleaved caspase-3 and BAX and up-regulated BCL-2 levels, while STAT3 levels were not significantly changed (Fig. 4C). Meanwhile, qRT-PCR results showed decreased mRNA expression of BAX and increased expression of BCL-2 (Fig. 4D). ELISA indicated that the inflammatory factors IL6 and TNF-α were considerably reduced in the supernatant of LPS-challenged HK2 cells after the knockdown of TASL expression (Fig. 4E). Consistent results were shown by qRT-PCR, where the mRNA levels of TNF-α and IL6 were also reduced (Fig. 4F). In addition, it was observed that LPS treatment notably elevated the percentage of apoptotic cells in TUNEL staining, and silencing of TASL expression counteracted this up-regulation (Fig. 4G). Thus, TASL is involved in LPS-induced renal tubular epithelial cell injury, and inhibition of its expression could curb apoptosis and inflammation.

In LPS-induced HK2 cells, Western blot showed that knockdown of STAT3 down-regulated STAT3, TASL, cleaved caspase-3, and BAX protein levels, accompanied by an increase in BCL-2 protein levels (Fig. 5A). qRT-PCR highlighted that mRNA expression of STAT3, TASL, and BAX was notably reduced, accompanied by an increase in BCL-2 expression (Fig. 5B). ELISA showed that knockdown of STAT3 down-regulated the levels of inflammatory factors IL6 and TNF-α in the supernatants of LPS-induced HK2 cells (Fig. 5C). Their mRNA expression was also reduced, as evidenced by qRT-PCR assay (Fig. 5D). TUNEL staining showed that the knockdown of STAT3 in LPS-stimulated HK2 cells significantly reduced the percentage of apoptotic cells (Fig. 5E). The above results suggest that the knockdown of STAT3 can suppress apoptosis and inflammation in LPS-stimulated HK2 cells.

Functional rescue assays were performed to further investigate whether STAT3 affected apoptosis and inflammation through transcriptional regulation of TASL. Western blot results showed that the protein levels of STAT3, TASL, cleaved caspase-3, and BAX were elevated, while the BCL-2 protein level was reduced after ectopic expression of STAT3, indicating elevated cell apoptosis. In contrast, transfection of siTASL negated the elevated apoptosis caused by overexpression of STAT3 (Fig. 6A). Meanwhile, ELISA results indicated that inflammatory factors IL6 and TNF-α in the supernatants were notably increased in LPS-treated HK2 cells after overexpression of STAT3, while knockdown of TASL attenuated the worsening of inflammation. The qRT-PCR results found that in LPS-treated HK2 cells overexpressing STAT3, IL6, and TNF-α mRNA expression was down-regulated after silencing of TASL (Fig. 6B). In addition, flow cytometric results showed that boosted apoptosis was identified in LPS-treated HK2 cells overexpressing STAT3, while the apoptosis was curbed after the knockdown of TASL expression (Fig. 6C). Meanwhile, TUNEL staining showed that knockdown of TASL in LPS-induced HK2 cells overexpressing STAT3 significantly decreased the TUNEL-positive rate of the cells (Fig. 6D). Therefore, all the above results suggest that the transcriptional regulation of TASL by STAT3 affects apoptosis and inflammation in LPS-induced HK2 cells.