Inhibitory effect of a novel Curcumin derivative DMC-HA on keloid fibroblasts

DMC-HA is more effective than Curcumin in inhibiting keloid fibroblast proliferation

Vimentin and CD90 serve as valuable markers in differentiating fibroblasts and exploring their roles in diverse biological processes. In our study, we performed in situ immunohistochemical staining for Vimentin and CD90 on primary cultured keloid fibroblasts. As depicted in Figure 1A, the immunohistochemical staining revealed robust expression of Vimentin and CD90 primarily within the cytoplasm of keloid fibroblasts, a finding further corroborated by flow cytometry analysis.

Figure 1. Effects of DMC-HA and Curcumin on the cell viability of primary keloid fibroblasts. (A) Immunocytochemistry and flow cytometry revealed primary keloid fibroblasts expressed Vimentin and CD90 which were major markers of fibroblast; (B) Cell viability of primary keloid fibroblasts was assessed using the CCK-8 assay following treatment with DMC-HA and Curcumin; (C) IC50 of DMC-HA and Curcumin on primary keloid fibroblasts.

Subsequently, we subjected these keloid fibroblasts to DMC-HA and Curcumin treatments at varying concentrations (0, 5, 10, and 20 μM) for a duration of 72 h. The CCK-8 assay results, displayed in Figure 1B, indicate that both drugs demonstrated the ability to inhibit keloid fibroblast proliferation, with the inhibitory effect intensifying as drug concentration increased. However, when comparing equivalent concentration levels, it became evident that DMC-HA exhibited a significantly superior inhibitory effect on cell proliferation compared to Curcumin (P<0.05). Further analysis revealed that the IC50 value for DMC-HA was 6.02 μM, a significant improvement over Curcumin, which had an IC50 value of 16.02 μM (Figure 1C).

DMC-HA is more effective than Curcumin in inducing apoptosis of keloid fibroblasts

Following the determination of the IC50 value through the aforementioned CCK-8 assay, we proceeded to assess the impact of DMC-HA on keloid fibroblast apoptosis. Keloid fibroblasts were subjected to treatments with varying concentrations of 5 and 10 μM of DMC-HA or Curcumin for a duration of 24 h, after which apoptosis was quantified using flow cytometry. As depicted in Figure 2A, the flow cytometric analysis consistently demonstrated a significantly higher rate of apoptosis in keloid fibroblasts when exposed to DMC-HA compared to Curcumin, across equivalent concentrations of 5 and 10 μM (P<0.05). Notably, Curcumin concentrations below 5 μM failed to induce substantial apoptosis in keloid fibroblasts (P>0.05).

Figure 2. Effects of DMC-HA and Curcumin on the cell apoptosis of primary keloid fibroblasts. (A) Cell apoptosis of primary keloid fibroblasts was assessed using the flow cytometry assay following treatment with Curcumin or DMC-HA; (B) The caspase-3 activity of primary keloid fibroblasts was detected after Curcumin or DMC-HA treatment.

To further support these findings, we conducted an assessment of caspase-3 activity, a crucial marker of apoptosis. The results were striking, indicating that 10 μM DMC-HA led to a significant 1.68-fold increase in caspase-3 activity when compared to the same concentration of Curcumin (Figure 2B). These results underscore the remarkable apoptotic-inducing potential of DMC-HA, particularly at the 10 μM concentration.

DMC-HA induces keloid fibroblast apoptosis through ROS

To investigate the impact of DMC-BH on ROS production, cells were pre-exposed to 5 and 10 μM DMC-BH for 24 h. In comparison to the control group, flow cytometry analysis revealed a noteworthy increase in ROS levels following DMC-HA pretreatment (P<0.05) (Figure 3A). Subsequently, Western blot analysis, conducted after a 24-hour DMC-HA intervention, demonstrated a substantial upregulation in intracellular mitochondrial apoptosis-related proteins, including cleaved caspase-3 (c.Casp.-3), cleaved PARP (c.PARP), Bax, and cytochrome C (Cyto C) (P<0.05) (Figure 3B).

Figure 3. Effects of DMC-HA on ROS production. (A) The impact of DMC-HA on ROS generation; (B) The effect of DMC-HA on the expression of c.Casp.-3, c.PARP, Bax, and Cyto C proteins; (C) NAC administration during DMC-HA intervention and its effect on ROS generation.
(D) NAC-mediated attenuation of cell apoptosis induced by DMC-HA; (E) NAC-induced alterations in the expression of c.Casp.-3, cleaved PARP, Bax, and Cyto C proteins in the presence of DMC-HA.

Upon the introduction of the ROS scavenger, NAC, a significant reduction in intracellular ROS levels induced by 10 μM DMC-HA in keloid fibroblasts was observed compared to the control group (Figure 3C). Additionally, pre-treatment with NAC ameliorated the apoptosis induced by DMC-HA (Figure 3D). Consistently, Western blot analysis revealed that pretreatment with NAC resulted in a reduction in the expression of c.Casp.-3, c.PARP, Bax, and Cyto C proteins, which had been upregulated by DMC-HA (Figure 3E).

DMC-HA inhibits the STAT3 pathway in keloid fibroblasts

We proceeded to investigate the potential involvement of the STAT3 signaling pathway in the inhibitory effect of DMC-HA on keloid fibroblasts. Previous studies have indicated that in vitro cultured keloid fibroblasts, similar to their normal fibroblast counterparts, either do not express phosphorylated STAT3 (p-STAT3) or express it at very low levels. However, the area surrounding keloid lesions exhibits heightened expression of IL-6, which contributes to keloid formation by inducing STAT3 phosphorylation in fibroblasts.

Consequently, upon the introduction of exogenous IL-6 to keloid fibroblasts, we observed a substantial increase in p-STAT3 levels, with the peak occurring at 15 min post-intervention (Figure 4A). Subsequent addition of DMC-HA resulted in a reduction in p-STAT3 levels induced by exogenous IL-6 in keloid fibroblasts, as evidenced by Western blot analysis (P<0.05) (Figure 4B). These findings suggest that DMC-HA may manifest its anti-scar effects through the inhibition of the STAT3 signaling pathway.

Figure 4. Effects of DMC-HA on STAT3 pathway. (A) p-STAT3 expression after exogenous IL-6 intervention; (B) p-STAT3 expression after exogenous IL-6 combined with DMC-HA intervention.

Materials

Keloid tissue specimens were acquired from patients undergoing surgical removal. No interventions or treatments were administered to the patients before the surgical procedures, ensuring the preservation of comprehensive clinical data. All aspects of this research were conducted in compliance with the guidelines and regulations established by the Ethics Committee of Affiliated Kunshan Hospital of Jiangsu University (2023-03-058-K01). Moreover, informed consent was diligently obtained from all participating patients, emphasizing the ethical integrity of the study.

Primary keloid fibroblast culture

Under strict sterile conditions, a small piece of tissue is excised from the patient’s keloid site. The excised scar tissue is then transferred to a sterile petri dish and subjected to multiple rinses with physiological saline or PBS (#KGB5001, KeyGEN Biotechnology, Nanjing, China) in order to eliminate blood and impurities. Subsequently, sterile scissors and forceps are used to cut the tissue into approximately 1mm³-sized pieces. After the addition of 0.25% trypsin/EDTA (#BL501A, Biosharp, Anhui, China) for digestion for 30 min, the tissue was gently agitated in a constant-temperature water bath set at 37° C for 1-2 h to aid in the release of fibroblasts from the tissue matrix. After the digestion process, complete culture medium (#KGM12800S-500, KeyGEN Biotechnology, Nanjing, China) was added to halt the reaction. Subsequently, the suspension was centrifuged at 1500 rpm for 5 min collect the cells. The supernatant was carefully decanted, and the cells were then resuspended in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (#12484028, Gibco BRL, Grand Island, NY, USA). These cells were maintained in a controlled incubator at 37° C with a 5% CO₂ environment. The initial medium change occurred after 3 days, followed by subsequent changes every 3 days. Routine passage and digestion were carried out when the cell confluence reached 70%-80%. Cells from passages 3 to 6 were used in the experimental procedures.

Immunofluorescence staining

Keloid fibroblasts were initially cultured in a culture dish and allowed to adhere to the dish’s surface. Subsequently, the cells were fixed with 4% acetaldehyde, followed by two 5-minute PBS washes. To block non-specific binding, a 5% bovine serum albumin solution was applied for 30 min. A Vimentin (#AF1975, Beyotime Biotechnology, Shanghai, China) or CD90 (#AF1636, Beyotime Biotechnology, Shanghai, China) primary antibody (1:100) was added, and the cells were incubated for 1 hour, after which they were washed with PBS to remove any unbound primary antibodies. A FITC-labeled goat anti-rabbit IgG (#A0562, Beyotime Biotechnology, Shanghai, China) (1:500) solution was then added to the cells and allowed to incubate for 30 min. Following this, the cells were washed with PBS to remove unbound secondary antibodies. Next, the nuclear stain DAPI (#C1005, Beyotime Biotechnology, Shanghai, China) was applied as a counterstain, and a drop of the appropriate anti-fade agent, Husolone, was added to the glass cover slip. The cover slip was then placed over the cells and secured using a blocking agent. Subsequently, a confocal microscopy was employed to visualize the fluorescent signal within the cells and capture images.

Flow cytometry for Vimentin and CD90 detection

Cultured cells were harvested and incubated with FITC-conjugated CD90 (#bsm-30105M-FITC, Bioss Biotechnology, Shanghai, China) and FITC-conjugated Vimentin (ab128507, Abcam, Cambridge, MA, USA) at 4° C for 30 min. After two washes, the cells were analyzed using a BD FACS Calibur cytometer. Flow cytometry data were processed using Flowjo software.

CCK-8 method for detecting cell proliferation

Keloid fibroblasts were collected and cultured, and the cells were seeded into 96-well plates at a density of 5,000 cells per well. When the cells are attached and grown to 70-80% passage, different concentrations of 0, 5, 10, 20 μM DMC-HA and Curcumin were added to treat the cells, and continued to culture them in a 37° C, 5% CO₂ incubator for 24, 48 or 72h to observe the effect of Curcumin on cell proliferation at different time points. After the treatment, 10 μL of CCK-8 solution (#C0037, Beyotime Biotechnology, Shanghai, China) was added to each well, and then continue to incubate the cells for 1-4 h in a 37° C, 5% CO₂ incubator. An enzyme-linked immunosorbent assay (ELISA) reader was employed to measure the absorbance values at a wavelength of 450 nm for each well. Subsequently, perform data analysis and generate graphical representations depicting the correlation between absorbance values and the concentrations of DMC-HA and Curcumin.

Flow cytometry for detecting cell apoptosis

Keloid fibroblasts were treated with different concentrations of DMC-HA and Curcumin, and the cells were collected at 24 h. Cells were centrifuged using serum-free medium and cell density was adjusted to 1x10^6 cells/mL. 100 μL of cell suspension was added to the flow cytometry tube. 5 μL of Annexin V-APC was added to each flow cytometry tube and incubated in the dark at room temperature for 15 min. 5 μL of PI dye was added to each flow cytometry tube and mix gently. Within 10 min after adding the PI dye, the cell apoptosis rate was analyzed using flow cytometry.

ROS measurement

To assess intracellular ROS production in DMC-HA-treated cells, fluorescence-activated cell sorting (FACS) analysis was conducted. Cells were exposed to varying DMC-HA concentrations for 24 h, followed by staining with 5 μg/ml of 2’,7’-dichlorofluorescin diacetate (DCF-DA) for 30 min. Subsequently, the stained cells were analyzed using a Becton-Dickinson FACS Caliber flow cytometer (Becton-Dickinson, San Jose, CA, USA).

Measurement of caspase 3 activity

The activity of Caspase 3 was assessed using a Caspase Activity Detection kit (Nanjing Kaiji Biotechnology, China) following the manufacturer’s instructions. Cell lysis was performed using the colorimetric buffers provided in the Caspase Activity Detection kit. Subsequently, proteins were pelleted by centrifugation at 12,000 × g for 10 min at 4° C and quantified using the BCA Protein assay kit (Nanjing Kaiji Biotechnology, China). Next, 100 μg of protein was incubated with 5 μl of the caspase 3 substrate (Ac-DEVD-pNA) in a 96-well plate. Caspase 3 activity was determined spectrophotometrically at 405 nm.

Western blot analysis

After keloid fibroblasts were treated with different concentrations of DMC-HA for 24 h, the cells were collected and proteins were extracted using RIPA lysis buffer, and then the cell lysate was separated by centrifugation. Next, the protein concentration was determined using the BCA method. An equal amount of protein sample (such as 50 μg) was added to the SDS-PAGE gel and electrophorese at 140V voltage for 1.5h. The protein was transferred to the PVDF membrane and 300mA current was used for transfer for 1 h. After the transfer is completed, 5% skim milk was used to block the non-specific binding sites of the PVDF membrane, and then incubated with specific primary antibodies at 4° C overnight. The next day, the membrane is incubated with the corresponding secondary antibody, usually for 1-2 h at room temperature. Finally, signals were detected with enhanced chemiluminescence (ECL) reagents, and images were acquired using a chemiluminescence imaging system. Analyze protein expression levels and perform quantitative and statistical analysis of experimental results as needed.

Statistical analysis

Data analysis was performed using SPSS 21.0 statistical software. In the comparison between multiple groups of data, one-way analysis of variance (ANOVA) was performed, while the t test was used for the comparison of two groups of experimental data. Set the test significance threshold α to 0.05.