2.1 Effects of Pae on cell viability of SZ95 sebocytes

We first evaluated the cytotoxicity of Pae in cultured SZ95 sebocytes using Cell Counting Kit-8 (CCK-8) assay. By comparing the cell viability of untreated cells, we found that Pae at low concentrations (≤ 80 µM) for 6 h treatment exerted no cytotoxic effect on SZ95 sebocytes (Fig. 1B). Consistent with CCK-8 results, Pae did not cause significant changes in cellular morphology at low treatment concentrations (≤ 80 µM). When SZ95 sebocytes were exposed to high concentrations (≥ 160 µM), pronounced cellular disruption was evident, manifested by cell shrinkage, disorganized intracellular contents, and compromised intercellular contact regions (Fig. 1C). Furthermore, our findings showed that IGF-1 alone or in combination with Pae at doses up to 80 µM did not caused any cytotoxicity on SZ95 sebocytes (Fig. 1D). As a result, the optimal concentrations of 80 µM Pae and 100 nM IGF-1 were chosen for further experiments in SZ95 sebocytes.

Fig. 1

Effects of Pae on cell viability of SZ95 sebocytes. A Chemical structure of Pae. B Various doses of Pae (0, 10, 20, 40, 80, 160 and 200 µM) were applied to SZ95 sebocytes for 6 h. Cell viability was determined by CCK-8. C Representative morphology of cells with or without Pae treatment after IGF-1 stimulus was observed under microscopy. Magnification, × 200. D Cells were incubated with 10, 20, 40 and 80 µM Pae alone or in combination with 100 nM IGF-1, followed by CCK-8 determination. Data are shown as means ± SD, n = 6. *P < 0.05, ****P < 0.0001 vs. untreated cells

2.2 Pae suppressed cell proliferation and altered the phase distribution of cell cycle in SZ95 sebocytes

Following treatment with Pae, the colony formation of SZ95 sebocytes was detected by crystal violet assay. Microscopy revealed a notable reduction in cell proliferation in response to treatment with 80 µM Pae. The proliferative levels did not show a statistical significance between the untreated and IGF-1-treated cells, while Pae significantly suppressed cell proliferation following stimulation with 100 nM IGF-1 (Fig. 2A and B). To evaluate whether Pae induced inhibition of proliferation through cell cycle progression, flow cytometric analysis was performed. Our results indicated that Pae led to an increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the S phase, while the phase distribution in G2/M remained unaffected by Pae treatment. These findings demonstrate that Pae could impede cell cycle progression by inducing cell arrest in the G0/G1 phase, thereby hindering the transition from G1 to the S phase in SZ95 sebocytes. Treatment with IGF-1 alone increased the cell population at G0/G1 phase, while reducing the proportion in the S and G2/M phases compared to untreated cells. Interestingly, Pae decreased the cell cycle distribution at the G2/M phase in IGF-1-treated SZ95 sebocytes, indicating a collaborative effect of IGF-1 and Pae in inducing cell cycle arrest (Fig. 2C and D).

Fig. 2

Effects of Pae on proliferation, cell cycle distribution and apoptosis in IGF-1-treated SZ95 sebocytes. After exposure to 100 nM IGF-1 for 1 h, cells were treated with 80 µM Pae for 6 h and then incubated in fresh medium for 24 h. A Cell proliferation was determined by crystal violet staining. Scale bars = 200 μm. B The optical density (OD) values of crystal violet were measured using a microplate reader at 590 nm. C Cell cycle progression was detected by flow cytometry using the PI/ RNase assay. G0/G1, interphase for DNA synthesis. S, DNA synthesis phase. G2/M, mitosis phase. D Cell cycle phase distribution (%) in SZ95 sebocytes treated with IGF-1 or Pae. E Representative morphology of apoptotic cells determined by AO/EB staining. Scale bars = 100 μm. F Cell apoptosis was assessed using the Annexin V/FITC assay. G The proportions of apoptosis include early and late apoptotic sebocytes. Data were expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. untreated cells. #P < 0.05, ###P < 0.001, ####P < 0.0001 vs. IGF-1-treated cells

In acne lesions, the augmentation of proliferative processes in sebaceous gland cells leads to an increase in the total cell population, thereby resulting in a corresponding rise in the quantity of lipids synthesized [17]. IGF-1 is considered as a mitogen that is essential for the proliferation and differentiation of epidermal cells. However, there is still controversy regarding the relationship between IGF-1 and sebocyte proliferation in vitro. Insulin and IGF-1 have been found to increase cell proliferation in rat preputial sebocytes [18]. Exposure to 1 µM insulin for 24 h stimulated the growth and proliferation of sebocytes, while another study conducted by Mirdamadi et al. [7]. reported that treatment with 1 µM insulin and 0.1 µM IGF-1 for 24 h resulted in a decrease in DNA replication in cultured SZ95 sebocytes. In this study, we found that the colony formation of SZ95 sebocytes was not affected compared to untreated cells after treatment with 100 nM IGF-1 for 24 h. These findings suggest that the effects of insulin and IGF-1 on sebocyte growth and replication may vary depending on the specific experimental conditions and cell models used.

However, flow-cytometric results indicated that IGF-1 blocked the phase transition from G0/G1 to S, suggesting that IGF-1 could prevent cells from entering the mitotic process necessary for DNA synthesis. Therefore, we hypothesized that although IGF-1 may hinder sebocyte proliferation by arresting cell cycle distribution, it required time for these effects to be reflected on the cellular proliferative processes.

2.3 Pae induced cell apoptosis in IGF-1-treated SZ95 seboctyes

To investigate the regulatory role of Pae in cell survival, the apoptotic process in SZ95 sebocytes was examined using the AO/EB staining kit. The AO dye labels the nuclei with green fluorescence, while EB only diffuses into cells with compromised membrane emitting red fluorescence. Cells treated with IGF-1 exhibited intense green fluorescence in the nuclei, with only a few apoptotic nuclei showing red fluorescence compared to untreated cells (Fig. 2E). In contrast, cells treated with Pae displayed early-stage apoptotic characteristics with pyknotic green nuclear staining and late-stage apoptotic cells with irregularly condensed orange-red nuclear staining. Apoptotic events were further assessed using Annexin V-FITC/PI staining. Our results indicated that IGF-1 reduced the proportion of apoptotic cells compared to untreated cells. In contrast, pre-incubation with Pae induced a significant increase of the apoptotic events following IGF-1 stimulation compared to the group treated with IGF-1 alone (Fig. 2F and G). Therefore, these findings suggest that Pae can reverse the inhibitory effect of IGF-1 on apoptosis in SZ95 sebocytes.

Apoptosis is a programmed cell death process under physiological conditions, which does not disrupt intracellular contents, thereby preventing the release of inflammatory mediators that could lead to tissue injury [19]. Isotretinoin, the most effective anti-acne agent, exhibits the strongest sebum-suppressive effect by inducing sebocyte apoptosis [3]. Therefore, promoting sebocyte apoptosis can not only reduce sebum secretion but also prevent excessive inflammatory responses. Consistent with previous studies [20], we found that IGF-1 exerted an inhibitory effect on apoptosis in SZ95 sebocytes. Pae has been proven to inhibit tumorigenesis by suppressing proliferation and inducting apoptosis in cancer cells [21]. Also, we found that Pae inhibited proliferation, arrested cell cycle progression and induced apoptosis in IGF-1-treated SZ95 sebocytes, suggesting that Pae has protective effects against IGF-I-induced sebocyte dysfunction with respect to cell growth and survival.

2.4 Pae ameliorated IGF-1-induced lipogenesis and downregulated lipogenic transcription factors in SZ95 sebocytes

We next sought to investigate the potential impact of Pae on IGF-1-induced lipogenesis. The lipid droplets were stained with Oil Red O (ORO) and Nile Red dyes to detect neutral lipids. Our results revealed an augmented intracellular lipid accumulation in SZ95 sebocytes treated with IGF-1, which was partially inhibited by Pae treatment (Fig. 3A–C). To understand the molecular basis underlying sebo-suppression induced by Pae, we examined the expression of the lipogenic transcription factor PPARγ through Western blotting. The protein levels of PPARγ were upregulated in IGF-1-treated cells, whereas this induction was significantly attenuated upon Pae administration (Fig. 3D and E). To further investigate the effects of Pae on sebum composition, we measured the levels of main neutral lipids, including Triglyceride (TG) and Free Fatty Acids (FFA). The levels of TG and FFA were increased by IGF-1, while these lipids were significantly reduced when Pae was administered (Fig. 3F and G). These findings indicate that Pae has the potential to inhibit the acne-like sebaceous lipogenesis pattern induced by IGF-1.

Fig. 3

Effects of Pae on lipogenesis and inflammation in IGF-treated SZ95 sebocytes. After pre-treatment with 100 nM IGF-1 for 1 h, cells were then incubated with 80 µM Pae for 6 h. A Intracellular lipids were visualized by Nile Red and ORO staining. Scale bars = 20 μm. B Relative fluorescence of Nile red staining (n = 3). C The OD values of supernatant ORO levels (%) were measured at 500 nm. D Western blot detection of PPARγ, with β-actin as a loading control. E Densitometry analysis of the PPARγ protein (n = 3). F and G Spectrophotometric analysis of TG and FFA levels (n = 5). H qRT-PCR analysis for the transcript levels of IL-6, IL-8 and TNF-α (n = 3). I ELISA analysis of pro-inflammatory cytokines (IL-6, IL-8 and TNF-α) levels in cell culture supernatant (n = 3). Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. untreated cells. ###P < 0.001, ####P < 0.0001 vs. IGF-1-treated cells

Previous research demonstrated a positive correlation between decreased sebum production and improvement in the severity of acne lesions [22]. PPARγ was upregulated in SGs derived from acne patients compared to those from healthy individuals [23], and its agonists can stimulate sebocyte differentiation and increase the rate of sebum excretion [24]. PPARγ activation serves as an essential co-factor for the induction of sebum production by IGF-1 [25]. In HFD-induced obese mice, Pae alleviated lipid accumulation in liver and adipose tissue by downregulating PPARγ [26, 27]. Our investigation supported prior findings by demonstrating a general rise in PPARγ expression in IGF-1-treated SZ95 sebocytes, while the administration of Pae effectively reversed the effects.

The acne pathogenesis involves excessive sebum secretion and intricate changes in lipid homeostasis [28]. PPARγ activation alters the lipid composition, particularly affecting the biosynthesis of TG and FFA in SZ95 sebocytes [29]. On the skin surface, lipases catalyze the hydrolysis of TG into FFA. Recent findings have proved that the presence of saturated fatty acids in FFA can induce lipid accumulation in cultured sebocytes [30]. Moreover, FFA can stimulate the inflammatory response in human sebocytes [31]. As a potent anti-inflammatory agent, the relationship between Pae and FFA-induced sebaceous inflammation is still unknown and requires further research.

2.5 Pae inhibited IGF-1-induced inflammation in SZ95 sebocytes

To evaluate the anti-inflammatory properties of Pae, the gene expression levels of L-6, IL-8, and TNF-α in SZ95 sebocytes were assessed using qRT-PCR after treatment with Pae. A significant reduction in the mRNA levels of IL-6, IL-8, and TNF-α was observed in Pae-treated SZ95 sebocytes compared to untreated cells. Furthermore, the increase in mRNA levels of IL-6, IL-8, and TNF-α caused by IGF-1 was significantly attenuated following treatment with Pae (Fig. 3H). Consistently, results from the ELISA analysis demonstrate that Pae was able to decrease the secretory protein levels of IL-6, IL-8, and TNF-α in IGF-1-treated sebocytes, further supporting the inhibitory effect of Pae on the inflammatory response activated by IGF-1. To further validate the findings, ELISA was performed to quantify the protein levels of IL-6, IL-8, and TNF-α. Consistently, the secretion of these cytokines was reduced in both normal and IGF-1-treated SZ95 sebocytes after exposure to Pae (Fig. 3I). These results indicate the inhibitory effect of Pae on the inflammatory response activated by IGF-1 in SZ95 sebocytes.

In addition to its lipogenic activities, IGF-1 plays a crucial role in the development of acne-associated perifollicular inflammation by upregulating the expression of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α [6]. The effect of Pae in disrupting LPS-stimulated renal inflammation has been previously demonstrated by reducing the release of IL-1β, IL-6 and TNF-α [32]. Our experiments used IGF-1 as a stimulator of cytokine synthesis, thereby inducing the transactivation of cytokine genes in acne pathogenesis and forming an in vitro model of sebaceous inflammation. In this study, we found that Pae abolished the secretion of IGF-1-induced pro-inflammatory factors from SZ95 sebocytes induced by IGF-1. Additionally, Pae downregulated the gene expression of IL-6, IL-8, and TNF-α, indicating its anti-inflammatory effect against IGF-1.

2.6 Inhibition of PI3K augments the attenuation of lipogenesis in Pae-treated SZ95 sebocytes

To investigate whether the effects of Pae in combating IGF-1-induced lipogenesis were mediated by the PI3K/ Akt/FoxO1 pathway, we examined the effects of Pae under the administration of PI3K inhibitor LY294002 (LY) in SZ95 sebocytes. Protein expression of p-Akt and p-FoxO1 were significantly upregulated by IGF-1, but downregulated by Pae and LY compared to untreated cells (Fig. 4A and B). Notably, the inhibitory effect of Pae was enhanced by the co-intervention of LY. However, there was no effect on the total levels of Akt and FoxO1. In addition, it has been shown that 14-3-3 inactivates FoxO1 by promoting the nuclear exclusion of p-FoxO1 [33]. Our result showed that protein levels of 14-3-3ζ were markedly decreased by Pae or co-treatment with LY, indicating that Pae might prevent the degradation of FoxO1 through inhibiting the activity of 14-3-3ζ. These results suggest that Pae could function as an antagonist of IGF-1 by inhibiting the phosphorylation of downstream PI3K/Akt/FoxO1 pathway.

Fig. 4

Pae mitigated IGF-1-induced lipogenesis in SZ95 sebocytes by targeting the PI3K/Akt/FoxO1 pathway. Following IGF-1 stimulation, cells were treated with 80 µM Pae alone for 6 h or in combination with 50 µM LY for 30 min. A and B Representative blots and quantitative analyses for p-Akt, Akt, p-FoxO1, FoxO1, and 14-3-3ζ expression in the untreated or IGF-1 and Pae-treated SZ95 sebocytes, with the protein intensities normalized to β-actin (n = 3). C Lipid droplets were visualized using Nile Red and ORO staining. Scale bars = 20 μm. D Relative fluorescence of Nile red staining (n = 3). E The OD values of supernatant ORO levels (%) were measured at 500 nm. F and G Quantitation of TG and FFA levels were spectrophotometrically measured (n = 5). H and I Representative blotting images and relevant quantitation of PPARγ normalized to β-actin (n = 3). Data were expressed as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. untreated cells. #P < 0.05, ##P < 0.01, ####P < 0.0001 vs. IGF-1-treated cells

Lipids staining was used to validate the findings above. Exposure to Pae, either alone or co-treated with LY effectively mitigated lipid overproduction induced by IGF-1 (Fig. 4C–E). Moreover, the combined treatment of Pae and LY substantially reversed the IGF-1-stimulated synthesis of TG and FFA (Fig. 4F, G), and attenuated the protein expression of PPARγ induced by IGF-1 (Fig. 4H). Collectively, these data suggest that the inhibitory effects of Pae on IGF-1-induced lipogenesis are, at least partially, attributed to the inactivation of the PI3K/Akt/FoxO1 pathway.

The transcriptional activity of FoxO1 relies on a nuclear-cytoplasmic transport system. IGF-1 triggers Akt-mediated FoxO1 phosphorylation through the PI3K signaling pathway, which ultimately results in FoxO1 degradation [7]. Studies have shown that Pae inhibited the PI3K/Akt pathway and restore FoxO1 expression, which may contribute to its anti-cancer effects [34, 35]. In our study, we found that Pae treatment significantly diminished the phosphorylation of Akt and FoxO1 induced by IGF-1. By binding to 14-3-3, p-FoxO1 undergoes conformational changes and travels from the nucleus to the cytoplasm for degradation [33]. Our results displayed that Pae decreased 14-3-3ζ expression in IGF-1-stimulated SZ95 sebocyte, suggesting that Pae can indirectly improve the nuclear retention of FoxO1. Nuclear retention of FoxO1 restrains the transcriptional activity of the PPAR family [36], which may explain the downregulation of PPARγ after Pae treatment. These findings imply that Pae may function as an inhibitor of the PI3K/Akt/FoxO1 pathway, thereby normalizing IGF-1-induced dysregulated lipogenesis. Moreover, we found an increase in TG and FFA production in response to IGF-1, while the overproduction of TG and FFA induced by IGF-1 was counteracted following LY incubation, suggesting that IGF-1 may disturb sebum composition through the PI3K pathway. Pae normalized sebaceous lipidomics by reducing the production of TG and FFA in IGF-1-treated SZ95 sebocytes through inhibition of the PI3K pathway, which may underpin the therapeutic effectiveness of Pae in acne therapy.

2.7 JAK2 inhibition blocked the anti-inflammatory effects of Pae in IGF-1-treated SZ95 sebocytes

Previous reports have implicated the protective effects of Pae on inflammatory progression by blocking the JAK2/STAT3 pathway in immune cells [12]. To explore whether Pae inhibited IGF-1-induced inflammation by modulating the JAK2/STAT3 pathway, we analyzed the impact of Pae under the intervention of the JAK2 inhibitor AG490 (AG) in SZ95 sebocytes. Our results indicated that Pae significantly decreased the expression of p-JAK2 and p-STAT3 induced by IGF-1 without effect on total levels of JAK2 and STAT3. The dephosphorylation of JAK2 and STAT3 induced by Pae was remarkably augmented in the presence of AG (Fig. 5A and B). To confirm our expectations, the levels of pro-inflammatory cytokines were analyzed by qRT-PCR. It was shown that co-incubation of Pae and AG significantly attenuated the mRNA levels of the IL-6, IL-8 and TNF-α in response to IGF-1 stimulus compared to the cells treated with IGF-1 and Pae (Fig. 5C–E). Consistent with this, the secreted IL-6, IL-8 and TNF-α protein levels were also decreased following Pae and AG treatment, as detected by ELISA (Fig. 5F–H). Overall, these findings suggest that targeting JAK2 can potentiate the anti-inflammatory effects of Pae.

Fig. 5

Pae alleviated IGF-1-induced inflammation in SZ95 sebocytes by inhibiting the JAK2/STAT3 pathway. Upon stimulation with IGF-1, cells were treated with 80 µM Pae for 6 h, or co-treated with 10 µM of AG for 2 h. A and B Representative blots for p-JAK2, JAK2, p-STAT3, and STAT3 are shown, along with quantification of protein intensities normalized to β-actin. C–E The mRNA levels of IL-6, IL-8 and TNF-α were evaluated using qRT-PCR. F–H Quantitative analyses of supernatant IL-6, IL-8 and TNF-α were measured using ELISA. Data were expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. untreated cells. #P < 0.01, ##P < 0.001, ####P < 0.0001 vs. IGF-1-treated cells

It has been verified that Pae protected against oxidative stress and alleviated TNF-α and IL-6 expression induced by oxygen-glucose deprivation in PC12 cells by modulating the JAK2/STAT3 pathway [37]. In SZ95 sebocytes, STAT3 activation by leptin contributes to the increased expression of proinflammatory cytokines including IL-6, IL-8, TNF-α and IL-1β, thereby promoting inflammation [10]. Consistent with previous studies, our results showed a decrease in the phosphorylated levels of JAK2 and STAT3 in Pae-treated SZ95 sebocytes, while JAK2 inhibition enhanced the inhibitory effect of Pae on attenuating the production of IL-6, IL-8 and TNF-α. Alternatively, STAT3 activation could result in an anti-inflammatory phenotype by increasing the expression of anti-inflammatory cytokine such as IL-10 in immune cells [38]. Previous studies reported that both the activation of IGF-1 receptor and IGF-1 exposure promote the binding and phosphorylation of JAK2 and STAT3 protein, thereby enhancing the immunosuppressive activity of murine T cell types [11]. In contrast, our analysis found that abolishment of the JAK2/STAT3 pathway or co-treatment with Pae attenuated the production of IL-6, IL-8 and TNF-α induced by IGF-1, suggesting that IGF-1 may stimulate inflammation in SZ95 sebocytes through activation of the JAK2/STAT3 pathway. Based on these findings, the roles of IGF-1 and JAK2/STAT3 in inflammatory processes can vary depending on the specific cell types and species involved. Further research is required to elucidate whether IGF-1 induces pro-inflammatory responses through the activation of JAK2/STAT3 pathway using in vitro models for acne.