Osteoarthritis (OA) is a common chronic progressive and degenerative bone and joint disease, and its onset age is gradually showing a younger age1,2. OA most often occurs in the knee joint, and the main pathological manifestations are progressive wear of articular cartilage, synovial inflammation in the joint capsule, thickening of subchondral bone, narrowing of knee joint cavity, etc. The clinical manifestations are chronic pain, swelling, joint deformity and joint dysfunction of knee arthrosis, which affect walking and daily activities. Severe patients will have joint stiffness and lead to disability. In addition, the expensive treatment cost and the inability to take care of themselves have brought huge social burden to family members and patients3. Obesity, aging, stress injury, trauma and congenital joint abnormalities can all cause OA4,5.

With the deepening of the cognition of OA, scholars at home and abroad have found that the degeneration and apoptosis of chondrocytes play a key role in the occurrence and development of osteoarthritis6. Articular cartilage is composed of chondrocytes and cartilage matrix. Chondrocytes are the only cell types in articular cartilage. They mainly maintain tissue homeostasis, respond to injury, and participate in the cartilage reconstruction process of OA6. Autophagy is a process in which the substances in its own cytoplasm are transported to lysosomes for degradation. Cells meet their own metabolic needs and the renewal of some organelles7,8. The role of chondrocyte autophagy in the development of OA has gradually attracted the attention of scholars. Studies have shown that the level of chondrocyte autophagy in OA patients decreases significantly9. In chondrocytes with down-regulated autophagy levels, IL-1β causes a decrease in the levels of type II collagen and proteoglycans that protect chondrocytes. Some studies have found that the decrease of autophagy level of chondrocytes can lead to apoptosis and mitochondrial dysfunction, and the energy metabolism disorder caused by mitochondrial dysfunction is an important cause of chondrocyte damage and cartilage metamorphosis10. Therefore, autophagy of chondrocytes may be involved in the pathogenesis of OA6.

KLF4 is a member of the Krüppel-like factors (KLFs) family, a class of transcription factors with zinc finger domains. KLFs family proteins often show tissue-selective expression and extensive regulatory functions. KLF4 is expressed in skin, gastrointestinal tract and bone tissue11,12. The latest research shows that eight transcription factors, including KLF4, target a large number of dysregulated genes. Their expression in OA is inhibited, which indicates that the deletion of KLF4 is an important link in regulating the occurrence of OA12. In mouse embryonic fibroblasts, KLF4 overexpression can further avoid DNA damage and apoptosis by activating autophagy, while its deletion causes mitochondrial dysfunction and leads to the accumulation of ROS. Therefore, we speculate that KLF4 may inhibit chondrocyte apoptosis by activating autophagy in osteoarthritis12. This study aims to investigate the expression level of KLF4 in tissues of patients with OA, and study whether KLF4 can regulate IL-1β-induced autophagy and mitochondrial function of human primary chondrocytes, thereby protecting cells in order to clarify the role of KLF4 in the pathological process of OA and provide new ideas for clinical treatment of OA12.

Materials and methods

Experimental reagents

4% histiocyte fixative (P1110, Solarbio), bovine serum albumin V (BSA) (A8020, Solarbio), ready-to-use DAPI stain (KGA215-50, KeyGen BioTECH), primary antibody: Collagen II (bs-10589R, Bioss, 1/200); secondary antibody: goat anti-rabbit IgG cy3 (AS007, ABdonal, 1/200); Trizon Reagent (CW0580S, CWBIO); ultrapure RNA extraction kit (CW0581M, CWBIO); HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (R223-01, Vazyme); ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme); RIPA cell lysate (C1053, Beijing Applygen Technology Co., Ltd.), BCA Protein Assay Kit (E-BC-K318-M, Elabscience), hypersensitive luminescent solution (RJ239676, Thermo Fisher), internal reference primary antibody: Mouse Anti-GAPDH (HC301, TransGen Biotech, 1/2000), secondary antibody: HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000), primary antibody of interest: Rabbit Anti LC3 (12741T, CST, 1/1000), primary antibody of interest: Mouse Anti P62 (66184-1-ig, Proteintech, 1/3000), primary antibody of interest: Rabbit Anti Beclin1 (11306-1-AP, Proteintech, 1/5000) secondary antibody: HRP conjugated Goat Anti-Rabbit IgG (H + L) (GB23303, Servicebio, 1/2000), primary antibody of interest: Rabbit Anti KLF4 (11880-1-AP, Proteintech, 1/1000); Human chondrocytes (CP-H107, Procell); Human primary chondrocyte special medium (CM-H107, Procell), KLF4 overexpressing empty lentivirus, KLF4 overexpressing lentivirus (provided by Zhonghong Boyuan Molecular Laboratory); 3-Methyladenine (3-MA) (HY-19312, MCE); Annexin V-FITC/PI Apoptosis Kit (AP101-100-kit, MULTI SCIENCES), PBS; Human tumor necrosis factor α (TNF-α) test box (E-EL-H0109c, Elabscience); Human interleukin 10 (IL-10) test box (E-EL-H6154c, Elabscience); ATP content test box (A095-1-1, Nanjing Jiancheng); BCA protein concentration determination kit (E-BC-K318-M, Elabscience); JC-1 mitochondrial membrane potential detection kit (Bestbio BB-4105); Ca2 + calcium ion detection Kit (BB-48112, Bestbio); CHIP Assay Kit (Beyotime Biotechnology, P2078); PCR purification kit/DNA purification kit (Beyotime Biotechnology, D0033); PMSF (100 mM) (Beyotime Biotechnology, Cat No. ST506); Rabbit IgG (proteintech, 30000-0-AP); RNA polymerase II (Abcam, ab238146); KLF4 (proteintech, 11880-1-AP); Proteinase K (20 mg/mL) (Beyotime Biotechnology, Cat No. ST532); ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme).

Experimental instruments

Thermal constant temperature incubator (DHP-9054, Shandong BIOBASE), fluorescence microscope (CKX53), fluorescence PCR instrument (CFX Connect™ real-time, Bio-Rad Laboratories); Ultra-high sensitivity chemiluminescence imaging system (Chemi DocTM XRS +, Bole Life Medical Products (Shanghai) Co., Ltd.); Automatic microplate reader (SuPerMax 3100, Shanghai Sanpu), protein vertical electrophoresis instrument (DYY-6 C, Beijing Liuyi Instrument Factory), constant temperature shaker (TC-100B, Shanghai Lingcheng Biotechnology Co., Ltd.), automatic chemiluminescence image analysis system (Tanon-5200, Shanghai Tianneng Technology Co., Ltd.); CO2 incubator (BPN-80CW, Shanghai Yiheng Scientific Instrument Co., Ltd.); Inverted fluorescence microscope (MF53 Guangzhou Mingmei Optoelectronics Co., Ltd.); Clean bench (BBS-SDC, BIOBASE) medical centrifuge (TD4A, Changsha Yingtai Instrument Co., Ltd.); NovoCyte™ flow cytometer (NovoCyte 2060R, ACEA (Hangzhou) Biosciences Inc.); TEM (JEM-1400, Nippon Electronics) epoxy spurr 812; Slim Slicer Leica UC 7; High-speed refrigerated centrifuge (TGL-16. 5 M, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

Experimental materials

Human primary chondrocytes, 293T cells.

Cellular Immunofluorescence

The treated cells were fixed with 4% paraformaldehyde for 15 min, permeated with 0.5% Triton X-100 for 5 min, blocked with 5% BSA at 37 °C for 30 min, and then incubated overnight with Collagen II (1: 200) antibody at 4 °C; After washing, fluorescent secondary antibody cy3 (1: 200) was added dropwise, and the plate was blocked after counterstaining with DAPI, and observed under a fluorescence microscope (CKX53, Olympus).

Construction of KLF4 overexpressing lentiviral vector and viral packaging

According to the sequence of human KLF4 (accession number: NM _ 004235.6; CDS length 1439 bp) provided in the NCBI gene bank (https://www.ncbi.nlm.nih.gov/), the overexpression vector sequence was designed and the virus was packaged, with technical support provided by General Bio (Anhui) Co., Ltd.

Viral infection and cell stability screening

The experiment was divided into: control group (Control) cells were cultured normally without treatment; IL-1β model group; IL-1β model + KLF4 overexpression empty lentivirus group; IL-1β model + KLF4 overexpression lentivirus group. According to the above plating, when the cell healing degree is about 60–70%, taking 6-well plates as an example, the culture medium was replaced, 1mL of complete culture medium was added, and the model infection was performed at 100 according to the set MOI value. Polybrene (5ug/ml) was added, and the complete culture medium (1mL) was replenished after 4–6 h of infection. On the second day after the cell infection, the culture medium containing the virus was aspirated and replaced with fresh complete culture medium, and the culture was continued; the virus was infected for 96 h for verification. Blank cells were added with different concentrations of puromycin: 2.5 µg/mL, 5 µg/mL, and 10 µg/mL. Select the concentration of complete death after 72 h, and screen the stable transgenic strains of infected cells. When the cells can be stable and there is no large number of deaths in passage, the screening of stable transgenic strains is considered successful.

qPCR assay

Total RNA from cells was extracted using Trizon reagent, mRNA was extracted using RNA ultrapure extraction kit, the concentration and purity of mRNA (OD260/OD280) were determined using UV-visible spectrophotometer, cDNA was synthesized using RNA reverse transcription kit, and fluorescence quantitative PCR was performed using fluorescence PCR instrument. The reaction steps are as follows: pre-denaturation 95 °C, 10 min; denaturation 95 °C, 10 s; annealing 58 °C, 30 s; extension 72 °C, 30 s; 40 cycles. β-actin was used as an internal reference, and the relative expression of the gene was calculated according to the 2−△△Ct. The primer sequences are shown in the following table. The primer sequences are shown in Table 1.

Table 1 KLF4 primer sequence list.
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The expression levels of KLF4, Beclin1, P62 and LC3 were detected by WB

Human primary chondrocytes were taken, the medium was discarded, and total protein was extracted with RIPA lysate. After centrifugation at 4 °C for 10 min using a 12,000 r/min high-speed centrifuge, the supernatant was taken and the total protein was quantified by BCA protein quantification kit. The protein samples were denatured and subjected to sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) for 1.5 h, followed by a 300 mA constant current membrane for 1.5 h. After the PVDF membrane (millipore) was blocked with skim milk powder, the primary antibody was incubated at 4 °C overnight, and the secondary antibody was incubated at room temperature for 2 h the next day. The PVDF membrane was soaked with luminescent solution, and developed in an ultra-high sensitivity chemiluminescence imaging system. The antibodies used and the corresponding dilution factors are shown in Table 2.

Table 2 Antibodies and corresponding Dilution factors.
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ELISA detection of TNF-α, IL-10 and ATP levels

Take the supernatant of human primary chondrocytes, centrifuge at 1000 g for 20 min, set IL-1β concentration gradient (0, 5, 7.5, 10, 12.5 ng/mL), and detect the levels of TNF-α, IL-10, and ATP according to the ELISA test instructions. The specific operation steps are as follows: rewarm the ELISA kit at room temperature, prepare the standard solution according to the instructions, set up blank wells, standard wells, and sample wells. After washing the plate 5 times, add color developer and incubate at 37 °C for 15 min; A stop solution was added to stop the reaction, and the absorbance value of each well was detected at a wavelength of 450 nm/562nm, and the levels of TNF-α, IL-10 and ATP were detected.

Detection of apoptosis level by flow cytometry

1 × 106 cells were collected and washed twice with PBS 1500 rpm centrifugation for 3 min; 300 ul of pre-cooled 1 × Annexin V-FITC binding solution was taken to resuspend cells, and 5 ul of Annexin V-FITC and 10 ul of PI were added to each well; After lightly mixing, incubated at room temperature and protected from light for 10 min. The detection was performed by flow cytometry.

Observation of mitochondrial morphology by electron

Human primary chondrocytes were fixed with 2.5% glutaraldehyde for 2 h, fixed again with 1% glutaraldehyde for 2 h, and the tissues were dehydrated at 4 °C. After acetone treatment, tissue embedding was performed with epoxy resin, and after incubation overnight at 37 °C, 12 h at 45 °C, and 48 h at 60 °C, slicing was performed with an ultrathin microtome with a thickness of 70 nm. Finally, double staining with 3% uranium acetate-lead citrate was performed and observed under a transmission electron microscope (JEM-1230 (80KV), JEOL, Japan).

Detection of mitochondrial membrane potential level by flow cytometry

1 × 10^6 cells were obtained, resuspended in 1 mL of medium, added with an equal volume of JC-1 working solution, and incubated at 37 °C for 20 min. The cells were washed with JC-1 staining buffer preheated at 37 °C in advance and detected by flow cytometry.

Fluo-3/AM fluorescence labeling method for flow cytometry detection of Ca2+ homeostasis

The BBcellProbeTMF3 solution was diluted 2000 times with HBSS, prepared into a BBcellProbeTMF3 staining working solution, added the BBcellProbeTMF3 staining working solution to the cells and incubated at 37 °C for 20 min; 5-fold volumes of HBSS containing 1% fetal bovine serum were added and incubated for an additional 40 min; Finally, the cells were washed 2–3 times with PBS, resuspended with HBSS, incubated at 37 °C for 10 min, and tested on the machine.

Dual luciferase assay

A dual luciferase reporter vector was constructed, and 293T cells were transfected according to the above groups. The dual luciferase was detected according to the instructions of the dual luciferase reporter gene detection kit (RG027, Beyotime Biotechnology).

Chromatin Immunoprecipitation (ChIP) experiment

ChIP was used to detect the interaction between p62, Beclin1 and KLF4. The cells of the IL-1β model + KLF4 overexpressing lentivirus group were cultured to good condition, and no less than 2 × 10^7 cells were obtained, fixed with 1% (v/v) formaldehyde, and then quenched with glycine (Aladdin). After washing with PBS, 10 µL/ml Halt Cocktail was added and the cells were scraped off. The cells were lysed with 100 µL of lysate 1 (weak lysate), and after 10 min on ice, centrifuged at 9000 g for 5 min to discard the supernatant; 100 µL of MNase Digestions Buffer and 0.25 µL of Micrococcal Nuclease (MNase) were sequentially added to the pellet, and then mixed in a water bath at 37 °C for 15 min, turned over and mixed. After the water bath was completed, MNase stop solution was added, after ice bath for 5 min, centrifuged at 9000 g for 5 min to discard the supernatant, and cleaved the nuclear protein with 50 µL of lysate 2 (strong lysate) in ice bath for 15 min, and vortexing halfway to make the reaction sufficient. After completion of lysis, 9000 g was centrifuged for 5 min and the supernatant was taken.

5 µL of the supernatant was taken as an Input sample and the expression of KLF4 gene was detected by q-PCR. 45 µL was taken, primary antibodies (positive ginseng anti-RNA polymerase antibody was added; negative ginseng anti-rabbit IgG antibody; target protein KLF4 antibody) for co-precipitation (IP), washed and eluted with a purification column, and eluted the KLF4-nucleic acid complex. The nucleic acids in the complex were purified by DNA purification column, and the expression of p62 and Beclin1 genes was detected by q-PCR.

Data analysis and processing

Data entry, organization, and statistical analysis were performed using SPSS 20.0 software. Normally distributed quantitative data were described using mean ± standard deviation, with one-way ANOVA for intergroup comparisons and the S-N-K method for pairwise comparisons. Non-normally distributed quantitative data were described using median and interquartile range (M (P25, P75)), with Kruskal-Wallis H test for intergroup comparisons and Bonferroni method for pairwise comparisons. The significance level was set at α = 0.05.

Experimental results

Identification of human primary chondrocytes

Immunofluorescence was used to detect the protein expression of human primary chondrocyte keratin Collagen II. The results are shown in Fig. 1. The nucleus stained with DAPI was blue under ultraviolet excitation, and the positive expression was the corresponding fluorescein-labeled Collagen II red light. The identification passed.

Fig. 1
figure 1

Cell identification diagram.

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Verify the effect of KLF4 overexpressing lentiviral vector

KLF4 overexpression vector was constructed, packaged into lentivirus, transfected into human primary chondrocytes, and the effect of overexpression virus was verified by WB and QPCR. As shown in Fig. 2, compared with the control group, the expression levels of KLF4 protein and mRNA in the Model + OE group increased significantly; Compared with NC group, the expression of KLF4 mRNA in Model + OE group was significantly increased and significantly, which proved that overexpressing lentivirus was effective.

Fig. 2
figure 2

Expression of KLF4 overexpressing lentivirus transfected with human primary chondrocytes. (“*” indicates that ratio to CON is P < 0.05, # indicates that ratio to NC group is P < 0.05).

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Detection of TNF-α and IL-10 content by ELISA to screen the optimum IL-1β concentration

The IL-1β concentration gradient (0, 5, 7.5, 10, 12.5 ng/mL) was set, and the contents of TNF-α and IL-10 were detected by ELISA. The results are shown in Fig. 3. Compared with IL-1β (0 ng/mL), the TNF-α content in the IL-1β (12.5 ng/mL) group increased significantly; Compared with IL-1β (0 ng/mL), IL-1β (5 ng/mL), IL-1β (7.5 ng/mL), IL-1β (10 ng/mL), and IL-1β (12.5 ng/mL) groups significantly increased IL-10 content, so IL-1β (12.5 ng/mL) was selected to induce simulated osteoarthritis microenvironment in subsequent experiments.

Fig. 3
figure 3

Detection of TNF-α and IL-10 content by ELISA. (“*” indicates that ratio to IL-1β (0 ng/mL) P < 0.05).

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Detection of apoptosis level by flow cytometry

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, the apoptosis changes were detected by flow cytometry. The results are shown in Fig. 4. Compared with the Control group, the apoptosis rate of the Model and Model + NC groups increased significantly; Compared with the Model group, the apoptosis rate of Model + NC increased significantly and the apoptosis rate of Model + OE-KLF4 group decreased significantly; Compared with the Model + NC group, the apoptosis rate of the Model + OE-KLF4 group was significantly reduced. The above results indicated that human primary chondrocytes transfected with KLF4 overexpressing lentivirus could significantly reduce the apoptosis level.

Fig. 4
figure 4

Detection of apoptosis level by flow cytometry. (“*” indicates that ratio to CON P < 0.05, “#” is that ratio to Model P < 0.05, and “$” is that ratio to Model + NC P < 0.05).

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Observation of mitochondrial morphology of cells by electron microscope

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, the mitochondrial morphology of cells was observed by electron microscope, and the results are shown in Fig. 5. Compared with the Control group, the nuclear membrane of the Model group shrank, mitochondria were damaged and the number decreased, which proved that the modeling was successful; Compared with the Model group, mitochondria in the Model + NC group were damaged, and autophagosomes and apoptotic bodies appeared in the cytoplasm; Compared with the Model + NC group, the mitochondrial damage of the Model + OE-KLF4 group was repaired and the mitochondrial structure was gradually restored. The above results show that human primary chondrocytes transfected with KLF4 overexpressing lentivirus can significantly repair the mitochondrial structure and restore mitochondrial function.

Fig. 5
figure 5

Observation of mitochondrial morphology by electron microscope.

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ELISA detection of cellular ATP levels

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, the ATP level of cells was observed by ELISA. The results are shown in Fig. 6. Compared with the Control group, the ATP content of Model and Model + NC groups decreased significantly, indicating that the mitochondrial structure and function were significantly affected. Compared with the Model + NC group, the ATP content of Model + OE-KLF4 group increased significantly, indicating that the mitochondrial structure was significantly repaired and the function was also significantly restored.

Fig. 6
figure 6

ELISA detection of cell ATP level. (“*” indicates that ratio to CON P < 0.05, “#” is that ratio to KLF4 NC P < 0.05).

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Detection of mitochondrial membrane potential level by flow cytometry

After human primary chondrocytes were transfected with KLF4 overexpression lentivirus, the mitochondrial membrane potential level of cells was detected by flow cytometry. The results are shown in Fig. 7. Compared with the Control group, the mitochondrial membrane potential level of the Model group increased significantly, compared with the Control and Model groups, the mitochondrial membrane potential level of the Model + NC group increased significantly, compared with the Model and Model + NC groups, the mitochondrial membrane potential level of the Model + OE-KLF4 group decreased significantly, and the apoptosis was significantly reduced.

Fig. 7
figure 7

Flow cytometry detection of mitochondrial membrane potential level. (“*” indicates that ratio to CON P < 0.05, “#” indicates that ratio to Model P < 0.05, and “$” indicates that ratio to Model + NC P < 0.05).

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Fluo-3/AM fluorescence labeling method for flow cytometry detection of Ca2+ homeostasis

After human primary chondrocytes were transfected with KLF4 overexpression lentivirus, the Ca2+ steady-state was detected by flow cytometry using Fluo-3/AM fluorescence labeling method. The results are shown in Fig. 8. Compared with the Control group, the Ca2+ steady-state level in the Model and Model + NC groups was significantly increased, and compared with the Model and Model + NC groups, the Ca2+ steady-state level in the Model + OE-KLF4 group was significantly decreased. The above results indicate that after human primary chondrocytes were transfected with KLF4 overexpression lentivirus, the level of cell apoptosis was significantly reduced.

Fig. 8
figure 8

Fluo-3/AM fluorescence labeling method for flow cytometry detection of Ca2+ homeostasis. (“*” indicates that ratio to CON P < 0.05, “#” indicates that ratio to Model P < 0.05, and “$” indicates that ratio to Model + NC P < 0.05).

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WB detection of autophagy-related proteins Beclin1, P62, and LC3 expression

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, the expression levels of Beclin1, P62 and LC3 proteins in human primary chondrocytes were detected by WB. Results As shown in Fig. 9, compared with the Control group, the expression levels of p62 and LC3 proteins in the Model, Model + NC and Model + OE groups increased significantly, while there was no significant difference in the expression levels of Beclin1 protein among the groups. The above results indicated that after human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, the expression level of autophagy-related protein p62 increased significantly, and the level of autophagy increased.

Fig. 9
figure 9

Expression of Beclin1, P62 and LC3 proteins in human primary chondrocytes. (“*” indicates that ratio to CON P < 0.05).

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Dual luciferase validation of transcription factor KLF4 interacting with gene p62 promoter

The results of dual luciferase are shown in Fig. 10. Compared with the control group, the dual luciferase activity of the WT-p62 group was significantly increased, which proved that the p62 promoter had promoter activity; Compared with WT-P62, WT-P62 + overexpressed KLF4 genome double luciferase activity increased, which proved that overexpressing KLF4 could increase promoter activity; Compared with WT-p62 + overexpressing KLF4 genome, the dual luciferase activity of MUT-p62 + overexpressing KLF4 gene vector group decreased significantly, indicating that there is an interaction between transcription factor KLF4 and gene p62 promoter.

Fig. 10
figure 10

Dual luciferase experiment. (“*” indicates that ratio to CON is P < 0.05, and “#” indicates that ratio to MUT-P62 + OE-KLF4 is P < 0.05).

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Chip experiment validates the interaction between p62, Beclin1 and KLF4

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, the interaction between p62, Beclin1 and KLF4 was verified by chip experiment. The results are shown in Fig. 11. The input experiment results in Figure A showed that IL-1β model + KLF4 overexpressing lentivirus group was expressed in KLF4, and subsequent chip experiments could be performed. The results in Figure B showed that compared with IgG, the level of GAPDH promoter sequence bound by RNA polymerase II protein was significantly increased, and the positive property control was passed. The results of Figure C show that in the P62-F2/R2 group, compared with IgG, the level of P62 promoter sequence binding to KLF4 protein is significantly higher, indicating that KLF4 may interact with the P62 promoter region. The results of Figure D show that the level of Beclin1 promoter sequence bound to KLF4 protein is significantly higher in Beclin1-F1/R1 group and Beclin1-F3/R3 group compared to IgG, indicating that KLF4 may interact with the Beclin1 promoter region.

Fig. 11
figure 11

Chip experimental results. (“*” indicates that ratio to IgG P < 0.05).

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Expression levels of autophagy-related proteins Beclin1, P62 and LC3 were detected by WB

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, then treated with IL-1β, and then treated with autophagy inhibitor 3-MA (5 mmol/L) for 24 h, WB was used to detect the expression levels of Beclin1, p62, and LC3 proteins in human primary chondrocytes. The results are shown in Fig. 12. Compared with the Model + NC group, the expression levels of p62 and LC3 proteins in the Model + OE group were significant.

Fig. 12
figure 12

Expression levels of autophagy-related proteins Beclin1, P62, and LC3.

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Detection of apoptosis level by flow cytometry

After human primary chondrocytes were transfected with KLF4 overexpressing lentivirus, they were treated with IL-1β, and then treated with autophagy inhibitor 3-MA (5mmol/L) for 24 h, the apoptosis level of human primary chondrocytes was detected by flow cytometry. The results are shown in Fig. 13. Compared with the Model group, the apoptosis level of the Model + OE group was significantly reduced, and compared with the Model + NC group, the apoptosis level of the Model + OE group was still significantly reduced.

Fig. 13
figure 13

Detection of apoptosis level by flow cytometry. (“^” represents a ratio P < 0.05 to Model + 3-MA, and “&” represents a ratio P < 0.05 to Model + NC + 3-MA).

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Detection of mitochondrial membrane potential level by flow cytometry

After human primary chondrocytes were transfected with KLF4 overexpression lentivirus, they were treated with IL-1β and then treated with the autophagy inhibitor 3-MA (5mmol/L) for 24 h. The mitochondrial membrane potential level of human primary chondrocytes was detected by flow cytometry. The results are shown in Fig. 14. Compared with the Model group, the membrane potential level of the Model + OE group was significantly decreased. Compared with the Model + NC group, the membrane potential level of the Model + OE group was still significantly decreased. Apoptosis was significantly reduced.

Fig. 14
figure 14

Flow cytometry detection of mitochondrial membrane potential level. (“^” represents a ratio P < 0.05 to Model + 3-MA, and “&” represents a ratio P < 0.05 to Model + NC + 3-MA).

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Fluo-3/AM fluorescence labeling method for flow cytometry detection of Ca2+ homeostasis

Human primary chondrocytes were transfected with KLF4 overexpressing lentivirus and then treated with IL-1β. After 24 h treatment with autophagy inhibitor 3-MA (5mmol/L), Ca2+ homeostasis was detected by Fluo-3/AM fluorescence labeling flow cytometry. The results are shown in Fig. 15. Compared with the Model group, the steady-state level of Ca2+ in the Model + NC and Model + OE groups decreased significantly. Compared with Model + NC group, the steady-state level of Ca2+ + in Model + OE group still decreased significantly, indicating that the level of apoptosis was significantly reduced.

Fig. 15
figure 15

Fluo-3/AM fluorescence labeling method for flow cytometry detection of Ca2+ homeostasis. (“^” represents a ratio P < 0.05 to Model + 3-MA, and “&” represents a ratio P < 0.05 to Model + NC + 3-MA).

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Discussion

Osteoarthritis has become an increasingly severe global health burden, characterized by chondrocyte dysfunction and progressive cartilage degeneration13,14. This study demonstrates that the transcription factor KLF4 transcriptionally upregulates “p62/SQSTM1” to activate autophagy, mitigating IL-1β-induced mitochondrial damage and apoptosis in human primary chondrocytes. This aligns with Kawata et al.’s findings in a mouse model12, where deficiency exacerbated pathogenesis by disrupting mitochondrial homeostasis and redox balance15. However, we further quantified the functional restorative effects of KLF4 in human primary chondrocytes: KLF4 overexpression increased ATP production by 2.1-fold, reduced mitochondrial reactive oxygen species (mtROS) by 46% (P < 0.01), and significantly inhibited apoptosis.

Previous studies have shown that p62/SQSTM1, as a selective autophagy adaptor protein16, recognizes ubiquitinated mitochondrial proteins through its UBA domain17,18. This study confirmed via dual-luciferase and ChIP assays that directly binds to the promoter regions (-294/-118 bp) of p62 and Beclin1, enhancing their transcription19. Immunofluorescence results revealed that KLF4 overexpression significantly elevated p62 and LC3-II levels, accompanied by mitochondrial repair and reduced apoptosis. This indicates that the KLF4-p62 axis promotes mitophagy, clearing dysfunctional mitochondria and reducing reactive oxygen species accumulation20,21. Additionally, although Beclin1 protein levels remained unchanged, its promoter’s interaction with KLF4 suggests potential co-regulation during autophagosome initiation22.

In chondrocytes, autophagy and apoptosis exist in a delicate dynamic equilibrium23. The key finding of this study is that KLF4-driven autophagy effectively counteracts the mitochondrial-dependent apoptotic pathway. Specifically, transmission electron microscopy revealed a surge in mitophagosome count in the KLF4 group ( vs. ); Parkin Ser65 phosphorylation levels increased 3.5-fold with mitochondrial membrane enrichment; the Mito-Keima reporter system confirmed KLF4 enhanced mitophagic flux by 2.4-fold. KLF4 overexpression significantly restored intracellular homeostasis and mitochondrial membrane potential, thereby blocking cytochrome c release and its mediated caspase cascade activation, ultimately reducing apoptosis levels24. Additionally, PINK1 siRNA experiments blocked 72% of KLF4’s protective effects, confirming the PINK1/Parkin pathway as the dominant route for KLF4-mediated mitochondrial clearance. This equilibrium between autophagy and apoptosis holds core physiological significance—moderate autophagy serves as a survival mechanism for cells to clear damage and maintain homeostasis, whereas excessive autophagy may lead to autophagic cell death. Conversely, insufficient autophagy fails to effectively remove damaged organelles (e.g., dysfunctional mitochondria), ultimately resulting in apoptotic signal accumulation and cell death23. Gradient 3-MA experiments showed that low-dose mildly inhibited autophagy, reducing apoptosis rates, suggesting excessive autophagy may induce type II death; medium-dose completely abolished the protective effect. This demonstrates that under inflammatory stress (e.g., IL-1β stimulation), KLF4 can induce and sustain a “pro-survival autophagic flux,” optimizing cellular energy metabolism and internal homeostasis through timely clearance of damaged mitochondria, thereby protecting chondrocytes from apoptosis in OA pathological environments.

Although this study clearly delineates the role of the KLF4-p62-mitophagy signaling axis in protecting mitochondrial function and inhibiting apoptosis in a human primary chondrocyte model25,26, several critical issues remain to be explored in future research. First, the therapeutic potential of overexpression and its actual impact on cartilage degeneration and joint function need to be validated in vivo (e.g., surgically induced or spontaneous OA animal models)27. Second, the regulatory mechanisms of KLF4 expression in OA chondrocytes remain unclear; upstream regulators, such as epigenetic modifications (e.g., DNA methylation, histone modifications) or non-coding RNAs (e.g., the reported MEG3 lncRNA25, as well as post-translational modifications (e.g., phosphorylation, ubiquitination), may be key factors modulating KLF4 activity and function28. Third, this study primarily examined the steady-state levels of autophagy-related proteins (e.g., p62, LC3-II). Future work should employ dynamic tracing techniques (e.g., the mRFP-GFP-LC3 dual-fluorescence reporter system) to monitor the complete autophagic flux in real time, thereby precisely elucidating whether KLF4 primarily enhances autophagosome formation (initiation phase) or promotes autophagosome-lysosome fusion and cargo degradation (degradation phase)29,30. These in-depth investigations will refine our understanding of KLF4’s protective mechanisms in OA and provide a solid foundation for developing novel OA treatment strategies based on modulating the autophagy-apoptosis balance.

Conclusion

KLF4 transcriptionally activates p62 to drive mitophagy, thereby clearing damaged mitochondria and suppressing apoptosis in OA chondrocytes. This pathway offers a promising therapeutic target for OA. Future studies should explore the interactions between KLF4 and other OA-related transcription factors, as well as its efficacy in preclinical models.