THP-1 was obtained from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences; Fetal bovine serum, 1640 medium from Gibco (Thermo Fisher Scientific, Inc.); PBS buffer, trypsin–EDTA and penicillin from Guangzhou Xinhe Technology Co., Ltd. IGF-1 and phorbol 12-myristate 13-acetate (PMA), Cell Counting Kit-8, mitochondrial membrane potential assay kit, ECL chemiluminescence reagent were purchased from MedChemExpress; GAPDH antibody, cytochrome c antibody, phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1) antibody and Parkin antibody was purchased from Affinity Biosciences; Bax antibody was purchased from Cell Signaling Technology, Inc.; caspase-3 antibody and Bcl-2 antibody were purchased from Abcam; bovine serum albumin, DAPI, Hoechst 33342 and Normal Goat Serum were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Annexin V-FITC/PI apoptosis assay kit was purchased from Elabscience; BCA kit, caspase-3 activity assay kit, mitochondrial isolation kit and horseradish peroxidase-labelled goat anti-rabbit IgG were purchased from Hangzhou Biyuntian Biotechnology Co., Ltd.

Human-derived THP-1 cells were grown and incubated in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% inactivated fetal bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO₂. THP-1 cells were treated for 48 h with 100 nmol/ml PMA to induce macrophages differentiation, in which the cells transition from suspended to adherent growth and from round to irregular. Then the cells were treated with different doses of PA (0–800 µmol/l) dissolved in bovine serum albumin.

The effect of IGF-1 on the activity of THP-1 macrophages was assessed by the Cell Counting Kit-8 assay (cat. no. HY-K0301; MedChemExpress). THP-1 macrophages were co-treated with IGF-1 with PA (cat. no. P0500; MilliporeSigma) at different concentrations (1.0, 1.5, 2, 2.5 ng/ml) for 24 h. Then 10 µl of cell counting kit-8 reagent was added to each well, incubated for 4 h and the OD value measured at 450 nm and plot the standard curve.

Total protein was extracted using lysis buffer including protease inhibitors (cat. no. HY-K1001; MedChemExpress). Macrophages were washed with PBS after being lysed in an ice bath for 30 min in cell lysis buffer. Collect the cells in a centrifuge tube and centrifuge at 12,000 × g for 30 min at 4°C. The supernatant is the total cell protein. Protein concentration was determined using a BCA protein assay kit (cat. no. P0011; Hangzhou Biyuntian Biotechnology Co., Ltd.). Using 10–15% SDS-PAGE, equal amounts of protein (20 or 30 µg) were resolved and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk for 1 h at room temperature followed by incubation with the appropriate primary antibody: Rabbit anti-Bax (1:1,000; cat. no. 41162S; Cell Signaling Technology, Inc.), rabbit anti-Bcl-2 (1:1,000; cat. no. ab32124; Abcam), rabbit anti-caspase-3 (1:1,000; cat. no. ab32042; Abcam), rabbit anti-GAPDH (1:1,000; cat. no. AF7021; Affinity Biosciences), rabbit anti-cytochrome c (1:1,000; cat. no. AF0146; Affinity Biosciences), rabbit anti-PINK1 (1:1,000; cat. no. DF7742; Affinity Biosciences) and rabbit anti-Parkin (1:1,000; cat. no. AF0235; Affinity Biosciences) overnight at 4°C. Following that, secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG) were incubated for 1 h at room temperature. Finally, the Bio-Rad gel detection system (Bio-Rad Laboratories, Inc.) was used to observe the fluorescence intensity detection bands and ImageJ software (version 1.8.0; National Institutes of Health) used to quantify their density, and the ratio of the target protein to the internal reference protein was used as expression level of the target protein.

Annexin V-FITC/PI cell apoptosis detection kit (cat. no. E-CK-A211; Elabscience) was used for the analysis of cell apoptosis (early + late apoptotic cells) in accordance with the instructions. The treated cells were digested with trypsin solution without EDTA, harvested, washed with PBS, resuspended in 500 µl of 1X Annexin V Binding Buffer; 5 µl of Annexin V-FITC and PI was added and incubated in the dark for 15 min at room temperature. BD Accuri C6 Plus cytometer (BD Biosciences) was used for data collection. Flow.JoX (version 10.0.7; flowjo.com) software was used for data analysis.

The activity of caspase-3 was assessed using the caspase-3 kit assay (cat. no. C1115; Beyotime Institute of Biotechnology). The cells were lysed after treatment and Ac-DEVD-pNA was used as a substrate for caspase-3. Caspase-3 activity and absorbance were measured at OD 405.

Cells were stained with Hoechst 33342 (cat. no. C0031; Beijing Solarbio Science & Technology Co., Ltd.) for 10 min at 37°C and then detected by fluorescence microscopy. When apoptosis occurs in cells, the nuclei of the apoptotic cells can be seen to be densely stained, or fragmented and densely stained.

Mitochondria were isolated from THP-1 macrophages according to the instructions of the Cellular Mitochondrial Isolation Kit (cat. no. C3601; Beyotime Institute of Biotechnology). THP-1 macrophages were digested with trypsin, harvested, resuspended in wash buffer, and centrifuged at 200 × g for 5 min at 4°C to collect the cellular sediment. Cells were resuspended in mitochondrial isolation reagent and incubated in an ice bath for 15 min, after which a glass homogenizer was used to obtain cell homogenates. The mitochondrial precipitates were separated by gradient centrifugation for 10 min at 1,000 × g and 4°C, then gently transfer the supernatant to another centrifuge tube. The supernatant was 3,500 × g again, centrifuged at 4°C for 10 min, and then transferred to a centrifuge tube. The collected supernatant was again centrifuged at 12,000 × g for 10 min at a 4°C. The cytoplasmic protein concentration was determined by the BCA method (Hangzhou Biyuntian Biotechnology Co., Ltd.).

The mitochondrial membrane potential was measured using the fluorescent dye JC-1 (cat. no. HY-K0601; MedChemExpress). THP-1 cells were seeded in a small confocal dish, the number of seeded cells being approximately 2×10⁵. They were cultured in an incubator at 37°C for 48 h to induce differentiation into macrophages, then treated with drugs for 24 h, stained with JC-1 for 20 min at 37°C, stained with DAPI (cat. no. C0065; Beijing Solarbio Science & Technology Co., Ltd.)-labelled nuclei for 10 min at 37°C and 5% CO₂, washed with PBS and 500 µl of 10% FBS RPMI-1640 medium added. The changes in cell membrane potential were observed under a laser confocal microscope (Carl Zeiss AG).

Mitochondrial ROS production was detected according to the mitochondrial ROS kit (cat. no. BB-46091, BestBio). The treated cells were digested with trypsin solution without EDTA, washed with PBS and incubated with mitochondrial reactive oxygen species staining solution at 37°C in the dark for 20 min. Pre-cooled 500 µl 1X PBS was added to the centrifuge tubes to resuspend the cells and immediately detected with a BD Accuri C6 Plus cytometer (version 1.0.23.1; BD Biosciences). Flow.JoX (version 10.0.7; flowjo.com) software was used for data analysis.

Treated cells were stained with green fluorescent MitoTracker Green FM (200 nM; cat. no. HY-135056; MedChemExpress) for 30 min, then fixed with pre-cooled methanol for 30 min, permeabilized with 0.1% Triton X-100 for 1 min and incubated blocked with 10% goat serum for 2 h at room temperature. Cells were incubated with LC3 primary antibody (1:1,000; cat. no. 3868; Cell Signaling Technology, Inc.) overnight at 4°C. After three washes with PBS, cells were incubated with secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (1:500; cat. no. S0006; Affinity Biosciences) for 1 h. After staining with DAPI for 10 min at 37°C, cells were observed using a confocal microscope (Carl Zeiss AG). Colocalization was assessed by line scanning using ImageJ software (version 1.8.0; National Institutes of Health) and line plots were generated using GraphPad (version 8.4.3; Dotmatics).

Mitophagy was detected by co-localization of mitochondria with lysosome. Cells were incubated with LysoTracker (100 nM; cat. no. L7528; Beijing Solarbio Science & Technology Co., Ltd.) and MitoTracker (300 nM; cat. no. 8778; Cell Signaling Technology, Inc.) for 30 min at 37°C and 5% CO₂. The cells were then washed with PBS. Cells were fixed with pre-cooled methanol in the dark and incubated in an ice bath for 30 min. Bright green fluorescence represented mitochondria and bright red fluorescence represented lysosomes. Cell images were acquired using a confocal microscope (Carl Zeiss AG).

SPSS 16.0 software (SPSS, Inc.) was used for statistical analysis of the data. Data normality was tested using the Kolmogorov-Smirnov test. Normally distributed quantitative variables are described as mean ± standard deviation. Normally distributed continuous variables were compared between groups using one-way analysis of variance. The LSD method was used for homogeneity of variance, and Games-Howell method was used for non-homogeneity of variance. While non-normally distributed variables were compared using the rank sum test. Qualitative variables were described by frequency (percentage) and χ² test was used for comparison between groups. Pearson's product-moment correlation coefficient was used to analyze the correlation between the indicators following normal distribution, and Spearman's rank correlation coefficient was used to analyze the correlation between the indicators not following normal distribution. P<0.05 was considered to indicate a statistically significant difference.

To assess the effect of IGF-1 on cell viability, Cell Counting Kit-8 assay was performed. Cell viability was examined at different PA concentrations (0–800 µmol/l) and 400 µmol/l was selected for subsequent experiments (Fig. 1A). Co-treatment of IGF-1 with PA at different concentrations (1.0, 1.5, 2, 2.5 ng/ml) for 24 h increased cell viability in a dose-dependent manner (Fig. 1B). Based on the results, 2.5 ng/ml IGF-1 was selected as the working concentration for subsequent experiments.

An increasing body of research suggests that PA triggers a number of relatively distinct mechanisms underlying apoptosis, including endoplasmic reticulum stress, ceramide and mitochondrial malfunction (22,23). However, the mechanism by which PA induces apoptosis in macrophages has not been fully elucidated. The present study investigated PA-induced apoptosis in macrophages. Apoptosis level was observed when cells were treated with PA at a concentration of 400 µmol/l. However, following co-treatment with IGF-1, PA-induced apoptosis was inhibited (Fig. 2). The results of Hoechst 33342 staining showed that compared with the control group, macrophages nuclei were deformed in the PA group and the apoptosis rate was obviously increased. Compared with the PA group, the IGF-1 and PA co-treated group reduced the apoptosis of macrophages (Fig. 2A and B). Flow cytometry analysis using Annexin V/PI double staining also confirmed that apoptosis was markedly increased in the PA group, while apoptosis was significantly reduced by co-treatment with IGF-1 (Fig. 2C-F). The above results suggested that IGF-1 attenuates PA damage to macrophages.

Caspase-3 is an indication of apoptosis in the late process. For the purpose of determining how IGF-1 affected the expression of caspase-3 in macrophages, a caspase-3 activity kit and western blotting was employed. The findings demonstrated that macrophages in the PA group had considerably higher caspase-3 expression (Fig. 3A and B). Caspase-3 activity and protein levels were considerably reduced with the co-treatment with IGF-1 compared with the PA group (Fig. 3C and D).

Bcl-2 and Bax genes are among the main genes involved in apoptosis. Studies have shown that Bcl-2 has a direct effect on mitochondrial membrane proteins and can directly prevent the opening of channels in the outer mitochondrial membrane, thus preventing the release of cytochrome c and achieving inhibition of apoptosis (24–26). Western blotting results showed that IGF-1 upregulated the expression of anti-apoptotic protein Bcl-2 and downregulated the expression of pro-apoptotic protein Bax compared with the PA group, while the Bcl-2/Bax ratio was significantly higher (Fig. 4).

The ability of IGF-1 ability to inhibit macrophages apoptosis was further studied. Previous research findings revealed that IGF-1 might regulate mitochondrial activity, which is crucial for cell survival (27,28). The current study examined the levels of mitochondrial apoptosis. According to the flow cytometry findings, macrophages in the IGF-1 group had considerably lower levels of mitochondrial ROS than those in the PA group (Fig. 5C and D). Changes in the potential of the mitochondrial membrane are related to ROS levels. Longer mitochondrial permeability transition pore openings might result in ROS bursts that disturb mitochondria at higher ROS levels (29). JC-1 staining demonstrated that PA treatment reduced the mitochondrial membrane potential of macrophages, but IGF-1 administration increased the membrane potential (Fig. 5A and B). Furthermore, the concentration of cytochrome c protein in the cytoplasm increased in the PA group at the same time and IGF-1 prevented the release of cytochrome c into the cytoplasm (Fig. 5E and F). According to the findings, IGF-1 prevented macrophages apoptosis via the mitochondrial pathway.

Previous studies have suggested that excessive mitophagy might result in mitochondrial damage (30–32). To evaluate whether IGF-1 protect mitochondrial damage based on mitophagy, the present study initially measured the engulfing mitochondria using co-localization of autophagosomes and mitochondria. The combined fluorescence signal of LC3 and MitoTracker showed that PA significantly enhanced the colocalization of autophagosomes with mitochondria (Fig. 6A and B). Under confocal microscopy, mitophagy and lysosome fusion was observed in the PA group. IGF-1 co-treatment significantly reduced mitochondrial lysosomal fusion, implying that IGF-1 inhibited mitophagy (Fig. 6C and D). The PINK1/Parkin pathway has recently been recognized as a crucial signaling pathway driving mitophagy in mammalian cells (33–35). Thus, PINK1 and Parkin expression levels were evaluated. Treatment with PA increased PINK1 and Parkin in the macrophages and the co-treatment with IGF-1 decreased the expression of both proteins (Fig. 6E-G). Taken together, these findings suggested that IGF-1 diminished PA-induced mitophagy.