Epigallocatechin-3-gallate (EGCG) reduced expression of Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) and Transforming Growth Factor-β1 (TGF-β1) in visceral pleural tissue cultures of patients with empyema

Data analysis

The data normality test uses the Shapiro Wilk test to determine the normality of the data. If the data were normally distributed, then proceed with the Levene test to test the homogeneity of the data. If the data were normally distributed and homogeneous, it was continued with the ANOVA test. This test was conducted to assess whether there was a difference in the mean expression of SCUBE3 and TGF-β1 between all groups. A multiple comparison test or Least Significant Difference (LSD) was conducted after the ANOVA test to compare the expression of SCUBE3 and TGF-β1 in one group and another. If the data were not normally distributed and not homogeneous, Kruskal Wallis and Mann Whitney tests were performed.

The mean SCUBE3 gene expression in 24 hours incubation

The results of the ANOVA test showed that there were no significant differences in all groups of 1×1 cm and 2×2 cm preparations (p=0.113) (p=0.678), respectively (Figures 1, 2). As for differences between groups, LSD analysis showed that there was a significant difference between the control with 0 hours incubation and the control with 24 hours incubation in the 1×1 cm preparations (p=0.025) (Figure 1).

Figure 1. Mean number of cells expressing SCUBE3 in 1×1 cm preparation of pleural tissue with 24 hours incubation period.

Notes: Significance values in all groups based on ANOVA test (p=0.113); “a” sign indicate significant differences between groups based on LSD comparison test (p<0.05), a=0.025.

Figure 2. Mean number of cells expressing SCUBE3 in 2×2 cm preparation of pleural tissue with 24 hours incubation period.

Notes: Significance values in all groups based on ANOVA test (p=0.678).

The mean SCUBE3 gene expression in 72 hours incubation

In samples incubated for 72 hours, the ANOVA test showed that there was no significant difference in the 1×1 cm preparation group (p=0.443) (Figure 3). A significant relationship was observed in the 2×2 cm preparation group (p=0.002) (Figure 4). LSD test showed a significant difference between the control with 0 hour incubation and the groups administered EGCG 50 μg and 100 μg in the 2×2 cm preparation (p=0.002) (p=0.001) respectively (Figure 4). Significant differences were also found between the control with 72 hours incubation and the groups administered EGCG 50 μg and 100 μg in 2×2 preparations (p=0.041) (p=0.008) respectively (Figure 4).

Figure 3. Mean number of cells expressing SCUBE3 in 1×1 cm preparation of pleural tissue with 72 hours incubation period.

Notes: Significance values in all groups based on ANOVA test (p=0.443).

Figure 4. Mean number of cells expressing SCUBE3 in 2×2 cm preparation of pleural tissue with 72 hours incubation period.

Notes: Significance in all groups based on ANOVA test (p=0.002); different “a,b,c”,…. indicate significant differences between groups based on LSD comparison test (p<0.05), SD: Standard Deviation, a=0.002, b=0.001, c=0.041, d=0.008.

The mean TGF-β1 gene expression in 24 hours incubation

The ANOVA test showed a significant relationship in the 1×1 cm preparation group (p=0.019) (Figure 5) and there was no significant relationship in the 2×2 preparation group (p=0.375) (Figure 6) LSD test showed significant differences between the control with 0 hours incubation and the control with 24 hours incubation (p=0.003) (Figure 5). Significant differences were also observed between the control group with 24 hours incubation and the group administered EGCG 50 μg and 100 μg in 1×1 cm preparation (p=0.016), (p=0.035) respectively (Figure 5).

Figure 5. Mean number of cells expressing TGF-β1 in 1×1 cm preparation of pleural tissue with 24 hours incubation period.

Notes: Significant values in all groups based on ANOVA test (p=0.019); different a,b,c,…. indicate significant differences between groups based on LSD comparison test (p < 0.05), SD: Standard Deviation, a=0.003, b=0.016, c=0.035.

Figure 6. Mean Number of Cells ExpressingTGF-β1 in 2×2 cm Preparation of Pleural Tissue with 24 Hours Incubation Period.

Notes: Significance values in all groups based on ANOVA test (p=0.375).

The mean TGF-β1 gene expression in 72 hours incubation

The ANOVA test showed there was no significant relationship in the group 1×1 cm preparation (p=0.65) (Figure 7). A significant relationship was observed in the 2×2 cm preparation group (p=0.014) (Figure 8). LSD test showed significant differences between the control with 0 hours incubation and the groups administered EGCG 50 μg and 100 μg in 2×2 preparations (p=0.005) respectively (Figure 8).

Figure 7. Mean number of cells expressing TGF-β1 in 1×1 cm preparation of pleural tissue with 72 hours incubation period.

Notes: Significance values in all groups based on ANOVA test (p=0.65).

Figure 8. Mean number of cells expressing TGF-β1 in 2×2 cm preparation of pleural tissue with 72 hours incubation period.

Notes: Significant values in all groups based on ANOVA test (p=0.014); different a,b,c,…. indicate significant differences between groups based on LSD comparison test (p<0.05), SD: Standard Deviation, a,b = 0.005.

Effect of EGCG on SCUBE3 expression in pleural tissue of empyema patients

This study found that pleural tissue from empyema patients were having SCUBE3 gene expression. This was determined by the presence of SCUBE3 gene expression in the control samples. To the knowledge of the researcher, there has not been any study discussing the expression of SCUBE3 in pleural tissue affected by empyema, although theoretically this gene can be expressed due to the inflammatory process. Several studies have shown that SCUBE3 is associated with the development of several human cancers like early stage lung cancer (Chou et al., 2013), osteosarcoma (Liang et al., 2015), liver cancer (Xu et al., 2022), breast cancer (Shen et al., 2024), and the tooth development phase (Huo et al., 2021). Based on these studies, it is possible that SCUBE3 expression can also increase in other types of diseases, such as inflammatory diseases caused by empyema infection.

To the best of our knowledge, research on SCUBE3 expression in patients with empyema has not been conducted. Several studies have shown that empyema indirectly affects SCUBE3 expression. This began with a study conducted by Fiorelli in 2016, who found that pleural fluid levels of MMP-1, MMP-8, and MMP-9 were higher in patients with empyema parapneumonic effusions. MMP-2 levels were higher in patients with effusion but without empyema (Fiorelli et al., 2016).

Regarding its relationship with SCUBE3, research conducted by Qin in 2021 showed that MMP-2 and MMP-9 are proteins known to show the highest correlation with SCUBE3. This research supports the finding that empyema patients with increased levels of MMP-2 and MMP-9 also show an increase in the expression of SCUBE3 (Huo et al., 2021). It is also possible that MMP-1 and MMP-8 which are also widely secreted in empyema patients can also be related to SCUBE3, this can be material for future research.

In this study, we found a significant difference between SCUBE3 expression in 1×1 cm 0-hour incubation control and the 24-hour incubation control. However, there was no difference between the 0-hour incubation control and the 72-hour incubation. This is possible because SCUBE3 expression is greater in the control with 24 hours incubation than 72 hour incubation. We have not found literature that specifically discusses the changes of SCUBE3 expression at certain times in pleural tissue culture. But, some study found that gene expression in tissue culture can change over time.

Research has demonstrated that gene expression is dynamic and can vary significantly depending on various factors, including the duration of culture, cellular environment, and specific treatments. Studies have shown that gene expression profiles can change at different time points during cell culture. For instance, one study observed significant transcriptional changes in cells treated with a carcinogen over time, with specific genes showing increased or decreased expression at various intervals from one hour to eight days post-treatment (Ohmori et al., 2022). Another study highlighted that the transcriptional profiles of human adipose-derived stem cells differ significantly between freshly isolated cells and those expanded in culture (passage 0 vs. passage 1). This indicates that the process of culturing cells can lead to substantial alterations in gene expression profiles (Januszyk et al., 2015). The results obtained in this study indicate that the expression of the SCUBE3 gene in the control samples decreases as the incubation time increases.

Regarding the effect of EGCG on SCUBE3, no studies have directly analyzed the relationship between the two. However, several studies have discussed the effect of EGCG on MMPs, a protein that is correlated with SCUBE3 (Huo et al., 2021). Research conducted by Garbisa et al. showed that EGCG can suppress the gelatinolytic activity of MMP2 and MMP9 (Garbisa et al., 2001). An explanation of this process was explained in Maeda-Yamamoto’s study which found that EGCG inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) and suppression of p38 activity which is required for the upregulation of MMP9 (Maeda-Yamamoto et al., 1999).

These findings suggest that the suppression of ERK phosphorylation by EGCG is involved in the decrease in MMP2 and MMP9 mRNA expression. Similar results were obtained by Cheng-hung and colleagues, who found that EGCG also reduced ERK phosphorylation and the levels of AP-1 and Sp1, leading to the downregulation of MMP2 and MMP9 (Chou et al., 2013).

The decreased expression of MMP2 and MMP9, which are proteins that correlate with SCUBE3, suggests that the decreased expression of MMP2 and MMP9 can also reduce the expression of SCUBE3. This supports the results obtained in this study, where researchers found that samples given EGCG at a concentration of 50 μg or 100 μg had a lower SCUBE3 expression than the control sample. In addition to the influence of EGCG as previously explained, a 72-hour incubation period resulted in lower SCUBE3 expression compared to a 24-hour incubation. This may be due to the fact that the longer EGCG is present in a tissue, the more it decreases the expression of SCUBE3 in that tissue. These results lead to a new assumption that SCUBE3 expression in pleural tissue samples from empyema patients may decrease over time, with a more significant reduction observed with EGCG administration.

This research found that the 2×2 cm samples treated with EGCG showed lower expression of SCUBE3 compared to the 1×1 cm samples. Based on the results obtained, we suspect that the empyema pleura samples have a better absorption capacity for EGCG in the 2×2 cm samples. This indicates that acquiring tissue to achieve the best potential of EGCG in pleural tissue is through culturing 2×2 cm samples.

Ideally, the optimal size for tissue culture involves small tissue fragments, since this facilitates enhanced perfusion and absorption. Larger tissue pieces possess a higher surface area, resulting in less efficient absorption compared to smaller fragments. On the other hand, if the tissue sample taken is too small, there are concerns that the tissue acquisition may not be optimal. Consequently, an optimal metric is essential for attaining better results in organ cultivation (Cheng et al., 2022).

Several studies indicates that the best size for culturing human pleural mesothelial cells and pleural fibroblasts is 8.5 mm (Metelmann et al., 2022). Therefore, for easier measurement, we standardized it to 1×1 cm. Meanwhile, for the 2×2 cm preparation, we were trying to expand the size to see if a larger size can still serve as a reference for making tissue preparations. Numerous studies indicate that bigger specimen sizes are susceptible to necrosis; however, our findings reveal that tissue samples measuring 2×2 cm did not exhibit necrosis. We suggest that utilizing a 2×2 cm preparation could encourage novel approaches in the examination of pleural tissue collection methodologies.

Effect of EGCG on TGF-β1 expression in pleural tissue of empyema patients

The increase in TGF-β1 expression in pleural control samples from empyema patients in this study is in line with the findings of several previous studies. Research conducted by Scott et al. in 2003 on rabbit-induced empyema found that there was an increase in TGF-β1 concentration in the pleural fluid of mice (Sasse et al., 2003). One year later this research was supported by Craig and friends who found that intrapleural injection of anti-TGF-β1 in the pleura of mice induced by empyema was known to inhibit the development of empyema and significantly reduce the process of rabbit pleural fibrosis (Kunz et al., 2004).

The opposite result was found by Samanta and colleagues in 2018, who observed an increase in TGF-β1 levels in mice with induced pleural empyema. The study found no significant difference in TGF-β1 concentration between the experimental and control groups (da Silva et al., 2018).

Differences in research results may be due to differences in sample types and research objects. Research conducted by Scott and Craig examined the concentration of TGF-β1 in rabbit pleural fluid and Samanta examined the concentration in mouse pleural fluid. Meanwhile, this study used human pleural tissue to observe the presence of TGF-β1 expression. The difference in sample types between pleural tissue in both humans and experimental animals may have affected the results obtained, although there are studies that show that there are highly homologous TGF-β1 amino acid sequences among mammalian species (Terrell et al., 1993).

We speculate that the increased expression of TGF-β1 in pleural tissue in this study occurred because of inflammation caused by empyema infection. This is based on the finding of TGF-β1 in mesothelial cells (Gerwin et al., 1987) alveolar macrophages (Assoian et al., 1987), lymphocytes, fibroblasts (Awad et al., 1998) and inflammatory cells in the stimulated pleural fluid. All these cell types are present in the visceral pleura and in some inflammatory exudates during early fibrotic formation in empyema (Martin et al., 2000). Previous studies that examined the relationship between TGF-β1 and pleural disease also suggested that the elevated TGF-β1 in pleural fluid is produced by inflammatory cells (Lee and Lane, 2001).

We found that the 1×1 cm control sample incubated for 24 hours had a higher expression of the TGF-β1 gene compared to the 1×1 cm control sample incubated for 72 hours. This resulted a significant difference between the 0-hour incubation control and the 24-hour incubation control. Researchers suspect that the reason for the significant increase in TGF-β1 expression after 24 hours of incubation is the same as that occurring in SCUBE3 expression. This leads to a new finding where the control samples of tissue culture incubated for 24 hours exhibit greater gene expression than those incubated for 72 hours.

This study also found that there was a decrease in TGF-β1 expression in 2×2 cm samples given EGCG 50 μg and 100 μg in the first 72 hours of incubation. In addition to the factor of EGCG administration, the TGF-β1 expression in the samples incubated for 72 hours was also lower than that in the samples incubated for 24 hours. The reason for this has been explained previously when discuss SCUBE3 expression.

To the best of our knowledge, no study has discussed the effect of EGCG on TGF-β1 in the pleural tissue. Research on this topic is often focused on lung fibrosis and cancer. For example, research conducted by Sriram and colleagues on lung fibrosis in bleomycin-induced mice showed that EGCG can inhibit fibroblast activation and collagen accumulation by inhibiting TGF-β1 expression. Based on these results, EGCG is considered to be an effective therapy for lung fibrosis. In an in vitro study, Sriram and colleagues also revealed that EGCG was able to reverse TGF-β1 induced proliferation and activation of fibroblasts (Sriram et al., 2015).

The same study was conducted by Tsai et al. in a rat lung fibrosis model. Tsai showed that EGCG decreased TGF-β1 expression, MMP-2, and MMP-9 levels, and also had anti-fibrotic effects. This study also found that EGCG reduced mortality; improved lung histological changes; reduced serum levels of TGF-β1, IL-6, IL-10, and TNFα; and reduced collagen deposition and fibroblast proliferation (Tsai et al., 2019).

Although the two studies were only on lung fibrosis, they support the results of this study, which showed that there was a decrease in TGF-β1 expression in the pleura of patients given EGCG as much as 50 μg and 100 μg EGCG in the first 72 hours of incubation. A possible mechanism to explain the inhibitory effect of EGCG on TGF-β1 activation is the suppression of various TGF-β1 activators such as inflammatory cytokines, MMPs, macrophages, and other inflammatory cells (Sriram et al., 2009). The authors also assume that such a mechanism is also involved in the reduction of TGF-β1 expression in pleural tissue.

Similar to the SCUBE3 expression, this study also found that the 2×2 cm preparation showed less TGF-β1 expression than the 1×1 cm preparation. These results further strengthen our previous hypothesis that the potential of EGCG is more potent in samples with a 2×2 cm preparation.

Limitations of this study

This study did not explore the presence of other profibrotic factors that could potentially influence the occurrence of pleural fibrosis such as matrixmetalloproteinase-1,2,8, and 9 (MMP-1, MMP-2, MMP-8, and MMP-9). 2. The researchers only studied the effects of EGCG on pleural tissue, it is preferable to perform a comparative trial of the effect of EGCG on the potential fibrosis in pulmonary tissue due to empiema.

Ethical and consent

The research was approved by the Universitas Andalas Ethical Clearance Committee (Protocol number: DP.04.03/D.XVI.XI/26/2024), and the gatekeeper’s consent letters were provided by Dr. M. Djamil of the Teaching Hospital on January 9, 2024 and is valid until January 2025. This research has been declared to be ethically appropriate in accordance to 7 (seven) WHO 2011 Standards, 1) Social values, 2) Scientific Values, 3) Equitable Assessment and Benefits, 4) Risks, 5) Persuasion/Exploitation, 6) Confidentiality and Privacy, and 7) Informed Consent, referring to the 2016 CIOMS Guidelines. This is as indicated by the fulfilment of the indicators of each standard. All the participants provided written informed consent.