The effect of Jian Gan powder on the proliferation, migration and polarization of macrophages and relative mechanism

Abstract

Context

Jian Gan powder (JGP) is a Chinese medicine compound comprised ginseng, Radix Paeoniae Alba, Radix Astragali, Salvia miltiorrhiza, Yujin, Rhizoma Cyperi, Fructus aurantii, Sophora flavescens, Yinchen, Bupleurum and licorice.

Objective

This study explored the inhibitory effects, polarization and potential mechanisms associated with JGP in macrophages.

Materials and methods

RAW264.7 cells were randomly divided into six groups for 24 h: control, lipopolysaccharide (LPS), overexpression, 1% JGP, 2% JGP, 4% JGP, 8% JGP and 16% JGP. The effects of JGP on RAW264.7 cell proliferation were assessed using colony formation assays and cell counting kit-8 (CCK-8) assays. The Transwell assay was used to evaluate its impact on RAW264.7 cell migration. Moreover, we analysed the interleukin-6 (IL-6)/signal transducer and activator of the transcription 3 (IL-6/STAT3) signaling pathway using quantitative real-time PCR and Western blotting. Furthermore, we examined the M1/M2 polarization levels.

Results

Unlike LPS stimulation, JGP serum treatment markedly suppressed macrophage proliferation and migration capacity, while STAT3 overexpression enhanced RAW264.7 cell proliferation and migration. JGP inhibited the proliferation and migration of RAW264.7 cells by attenuating the IL-6/STAT3 signaling pathway. Furthermore, it inhibited macrophage M1 polarization, promoting M2 polarization.

Discussion and conclusions

JGP effectively suppressed the cellular function of RAW264.7 cells by down-regulating the IL-6/STAT3 signaling pathway and modulating macrophage M1/M2 polarization. These findings provide valuable theoretical and experimental basis for considering the potential clinical application of JGP in the treatment of immune-mediated liver injury in clinical practice.

Keywords:

• JGP
• RAW264.7
• STAT3
• M1/M2 polarization

Introduction

Macrophages are a highly heterogeneous group of immune cells, predominantly originating from monocytes in response to local stimulation by growth factors and inflammatory factors (Epelman et al. 2014). Macrophages can be classified into two main subtypes: classically activated M1 macrophages and selectively activated M2 macrophages. Macrophage-mediated inflammatory responses are crucial in various disease processes, encompassing infections, tumor progression, tissue injury repair, fibrosis and autoimmune diseases (Mills et al. 2018; Shapouri-Moghaddam et al. 2018). M1 macrophages, induced by lipopolysaccharide (LPS) and interferon (IFN)-γ, promote inflammation through the secretion of IL-1β, IL-6, tumor necrosis factor (TNF)-α, interleukin (IL)-12 and IL-23. M2 macrophages, induced by IL-4 and IL-13, inhibit inflammation by secreting tumor growth factor (TGF)-β and IL-10 (Sica et al. 2015).

Regulation of macrophage-mediated inflammation plays a pivotal role in the immune modulation of traditional Chinese medicine. In this study, we focus on the effects of Jian Gan powder (JGP), a compound of traditional Chinese medicine consisting of ginseng, Radix Paeoniae Alba, Radix Astragali, Salvia miltiorrhiza, Yujin, Rhizoma Cyperi, Fructus Aurantii, Sophora flavescens, Yinchen, Bupleurum and licorice. JGP has been empirically prescribed for the treatment of chronic liver injury; its efficacy in addressing liver function damage, particularly in cases of chronic liver disease, such as chronic hepatitis B, has been well-established for more than two decades (Huang et al. 2006). Previous clinical investigations conducted by our research group have shown promising results in the treatment of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD) using JGP. These effects may be closely associated with their ability to improve intestinal microbiota dysbiosis (Zhao, Wang, et al. 2022). However, the impact of JGP on macrophages remains unclear. To address this knowledge gap, we explored how JGP influences macrophage growth and M1/M2 polarization using the mouse macrophage cell line RAW264.7. The results provide preliminary evidence suggesting the involvement of the IL-6/signal transducer and activator of the transcription (STAT3) signaling pathway in this intricate process.

Materials and methods

Cell culture and treatment

RAW264.7 cells were purchased from the American Type Culture Collection (Cat No. CL-0190, RRID: CVCL_0493, ATCC). Cells were cultured at 37 °C under 5% CO₂ and 95% air. RAW264.7 cells were maintained in a Dulbecco’s modified eagle medium (DMEM) (Cat No. C11965500BT, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (Cat No. 10270-106, Gibco, Waltham, MA) and 1% penicillin-streptomycin (Cat No. SV30010, Hyclone, Logan, UT). RAW264.7 cells were treated with different concentration gradients (1%, 2%, 4%, 8%, 16% and 32%) of JGP (Hai’an Traditional Chinese Medicine Hospital, Nantong, China) for 24 h before analysis.

Cell counting kit-8 (CCK-8) assay

RAW264.7 cells were seeded in 96-well plates (Cat No. 3599, Corning, NY) at 1 × 10⁴ cells (100 μL)/well. These cells were exposed to varying concentrations of drug-containing serum or control serum. After a 48 h incubation period, 10 μL of CCK8 solution (Cat No. 40203ES76, Yeasen Biotechnology, Shanghai, China) was added to each well, and the plates were cultured in a carbon dioxide incubator for 1 h. The optical density (OD) at a wavelength of 450 nm was then measured using an enzyme labeling instrument (Spark 10 M, Tecan, Männedorf, Switzerland).

Colony formation assay

The cells were seeded in six-well plates (Cat No. 3516, Corning) at a density of 500 cells per well and allowed to grow for two weeks. Subsequently, after completion of the culture period, the cells were fixed in pre-cooled methanol for 15 min and then stained with 0.1% crystal violet for 30 min. After staining, plates were washed with phosphate-buffered saline (PBS) (Cat No. 30256.01, Hyclone, Logan, UT). All experiments were conducted in triplicate to ensure the reliability of the results.

Migration assays

For cell counting purposes, 1x10⁵ RAW264.7 cells in 100 μL were evenly distributed in the Transwell chamber (Cat No. TCS013024, Jet Bio-Filtration Co., Ltd, Guangzhou, China). In the culture plate chamber, 500 μL of complete culture medium was added. Four distinct groups were established: a blank serum group, a 1% JGP drug-containing serum group, a 2% JGP drug-containing serum group, a 4% JGP drug-containing serum group, a 8% JGP drug-containing serum group and a 16% JGP drug-containing serum group. After 4 h of incubation, LPS was introduced into the upper and lower chambers at a concentration of 20 μg/L, and cells were kept in a 37 °C environment with 5% CO₂ for 24 h. Subsequently, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 30 min, and washed again 2–3 times with PBS. Finally, the cell images were captured using an inverted fluorescence microscope and quantified using ImageJ software.

Plasmid construction and lentivirus transfection

Pre-experiment of lentivirus transfection with RAW264.7 cells

Before conducting the main experiment, it is essential to determine the optimal multiplicity of infection (MOI) for lentivirus transfection and determine the most suitable transfection conditions. RAW264.7 cells were seeded in a 6-well plate when they reached a confluence level of approximately 30%. A suspension of RAW264.7 cells was prepared at 2 × 10⁵ cells/mL. Each well was then coated with 2 mL of this suspension, totaling 4 × 10⁵ cells per well in the 6-well plate. After a 12 h incubation period, lentivirus transfection was initiated.

Lentivirus transfection with RAW264.7 cells

RAW264.7 cells were seeded in a 6-well plate when they reached a confluence level of approximately 30%. These cells were prepared in a cell suspension at a concentration of 2 × 10⁵ cells/mL. The MOI value was determined based on pre-experimental results, and the appropriate sample quantity was calculated accordingly. Subsequently, lentivirus transfection and subsequent cell culture were performed in preparation for subsequent experiments.

Total RNA extraction and real-time PCR

After treating RAW264.7 cells in various experimental groups, total RNA was extracted using the Trizol method. Subsequently, cDNA was synthesized following the instructions provided by the reverse transcription kit. Quantitative PCR (qPCR) assessed mRNA expression levels using cDNA as the template. The relative expression of the target gene was determined using the 2^−ΔΔC t method, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as an internal reference. The primer sequences used for this analysis were synthesized by Shanghai Raw and Industrial Organisms. IL-6: 5′-CCACTTCACAAGTCGGAGGCTTA-3′ (forward) and 5′-CCAGTTTGGTAGCATCCATCATTTC-3′ (reverse). CD86: 5′-TGCACGTCTAAGCAAGGTCA-3′ (forward) and 5′-CCAGAACACACACAACGGTC-3′ (reverse). Mrc-1: 5′-GGATGGCTCTGGTGTGGAAC-3′ (forward) and 5′-TCTCGCTTCCCTCAAAGTGC-3′ (reverse). GAPDH: 5′-GTCTTCACCACCATGGAGAA-3′ (forward) and 5′-TAAGCAGTTGGTGGTGCAG-3′ (reverse).

Western blotting

The RAW264.7 cells were lysed using RIPA lysis buffer (Cat No. P0013C, Beyotime Biotech., Shanghai, China) containing 1% protease inhibitor (Cat No. ST505, Beyotime Biotech., China) after treatment with different concentrations of JGP. The protein concentration was quantified using a BCA kit (Cat No. P0009, Beyotime Biotech., China). Subsequently, the proteins were separated by electrophoresis on a 10% SDS-PAGE gel and then transferred to a PVDF membrane (Cat No. FFP22, Beyotime Biotech., China). The membrane was blocked with a TBST solution containing 5% skim milk powder for 1 h, followed by overnight incubation with the primary antibody p-STAT3 (Cat No. 9145 T, Cell Signaling Tech., Danvers, MA), STAT3 (Cat No. 9139, Cell Signaling Tech), TLR4 (Cat No. 14358, Cell Signaling Tech.), cleaved Caspase-3 (Cat No. 9664, Cell Signaling Tech.), Caspase-3 (Cat No. 9622S, Cell Signaling Tech.) (diluted 1: 1000). Subsequently, the membrane was incubated with a secondary antibody (Goat Anti-rabbit IgG: Cat No. A0208, Beyotime Biotech., China; Goat Anti-mouse IgG: Cat No. A0216, Beyotime Biotech., China) (diluted 1: 3000) for 2 h. Protein bands were visualized using an ECL chemiluminescence solution (Cat No. SB-WB011, Share-Bio, Hangzhou, China), and images were captured using a gel imager (Tianneng, Zhejiang, China). β-Tubulin (Cat No. BS1482M, Bio-World, Visalia, CA) was used to confirm equal protein loading. Finally, a quantitative analysis of the protein bands was performed using ImageJ software.

Statistical analysis

Data are presented as mean ± standard deviation and were graphed and analysed using GraphPad Prism version 8.0 software (GraphPad Software, Inc, La Jolla, CA). p < 0.05 was considered statistically significant.

Results

Determination of components in JGP using HPLC-UV

JGP content, comprising three batches (201130, 200829 and 200914), was determined using an Agilent 1260 Infinity II system equipped with a DAD detector (Agilent Technologies, Santa Clara, CA). Chromatographic separation was achieved using a Cosmosil 5C18-AR-II column (4.6 mm × 250 mm, 5 µm) with a gradient elution of solvent A (phosphoric acid water) and solvent B (phosphoric acid acetonitrile) according to the following conditions: 0–5 min, 90% A; 5–10 min, 90%–89% A; 10–13 min, 89% A; 13–15 min, 89%–87% A; 15–20 min, 87%–83% A; 20–30 min, 83% A; 30–31 min, 83%–80% A; and 31–43 min, 80% A. The flow rate was set at 1 mL/min, and the column temperature was maintained at 25 °C. The detector was programmed to measure wavelengths of 237 nm, 230 nm, 283 nm and 327 nm for detecting liquiritin, paeoniflorin, hesperidin and chlorogenic acid, respectively. Each sample was injected at a volume of 10 µL. As shown in Figure 1, the contents of chlorogenic acid, paeoniflorin, liquiritin and hesperidin in the three batches of preparations were found to be 4.544 mg/g, 18.074 mg/g, 3.392 mg/g and 3.653 mg/g, respectively.

Figure 1. Determination of components in JGP by HPLC-UV. (A) HPLC-UV chromatogram of chlorogenic acid, paeoniflorin, liquiritin and hesperidin standards. (B) HPLC-UV chromatogram of the JGP sample. (C) The content of chlorogenic acid, paeoniflorin, liquiritin and hesperidin in JGP. 1: chlorogenic acid; 2: paeoniflorin; 3: liquiritin; 4: hesperidin.

Effect of JGP on the proliferation and migration of RAW264.7 cells

To evaluate the effects of JGP on cell proliferation and migration in RAW264.7 cells, different concentrations of JGP, including 0, 1%, 2%, 4%, 8%, 16% and 32%, were incubated with RAW264.7 cells for 24 h. Figure 2(A) shows that JGP has a dose-dependent inhibitory effect on the proliferation of RAW264.7 cells, with IC₅₀ values of 5.326%, among which JGP ranged from 2% to 32% significantly suppressed the proliferation of RAW264.7 cells. Then we subjected cells to a 24-h incubation with LPS in combination with varying concentrations of JGP (1%, 2%, 4%, 8% and 16%). Cell proliferation was assessed using the CCK-8 method and a colony assay, while cell migration was evaluated using the transwell assay. As shown in Figure 2(B–F), compared to the LPS-stimulated group, treatment with 2%, 4%, 8% and 16% JGP serum significantly inhibited macrophage RAW264.7 cell proliferation and migration. This inhibition exhibited a distinct concentration-dependent gradient. Therefore, 2%, 4% and 8% JGP were selected as low, medium and high concentration for further research.

Figure 2. Effect of JGP on the proliferation and migration of RAW264.7 cells. (A) The cell viability of RAW264.7 cells was examined by CCK-8 assays. (B) RAW264.7 cells were treated with different concentrations of JGP. The proliferation of RAW264.7 cells was detected by the CCK8 assay. (C) The proliferation of RAW264.7 cells was evaluated using a cell colony formation assay. (D) Migration of RAW264.7 cells was evaluated using migration assays. (E,F) Quantification of the colony formation assay (E) and migration assay (F). The experiment was repeated in triplicate and the results are presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

STAT3 overexpression promotes the proliferation and migration of RAW264.7 cells

The IL-6/STAT3 signaling pathway is a key regulator of macrophage proliferation and migration (Zhao, Liu, et al. 2022; Mito et al. 2023). To investigate the molecular mechanisms underlying the inhibitory effects of JGP on RAW264.7 cell proliferation and migration, we established a RAW264.7 cell line that overexpressed STAT3 (Figure 3(A–E)). Our findings revealed that STAT3 overexpression significantly increased both the proliferation and migration capabilities of RAW264.7 cells (Figure 3(F–H)).

Figure 3. STAT3 overexpression promotes proliferation and migration of RAW264.7 cells. (A) Diagram of the construction pattern of the lentivirus vector. (B,C) Detection of transfection efficiency and determination of the final polybrene loading dose in 293T cells. (D) Construction of STAT3 cell line overexpression in RAW264.7 cells. (E) The expression of STAT3 in RAW264.7 cells observed by RT-PCR. (F,G) The proliferation and migration of RAW264.7 cells was evaluated using a cell colony formation assay and migration assays. (H,I) Quantification of the colony formation assay (H) and migration assay (I). The experiment was repeated in triplicate and the results are presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

JGP inhibits the proliferation and migration of RAW264.7 cells by inhibiting the IL-6/STAT3 signaling pathway

To explore the potential interaction between JGP and STAT3 in impeding the proliferation and migration of RAW264.7 cells, we initially exposed RAW264.7 cells to varying concentrations of JGP for 24 h. During this exposure, we observed a decrease in the expression of IL-6 mRNA in RAW264.7 cells (Figure 4(A)). Subsequently, we subjected LPS-stimulated RAW264.7 cells to different concentrations of JGP. The results revealed that LPS increased Toll-like receptor 4 (TLR4) expression in RAW264.7 cells. In contrast, the addition of JGP to LPS-stimulated RAW264.7 cells resulted in a reduction in TLR4 expression (Figure 4(B,C)). Further research revealed that JGP could reverse up-regulation of STAT3 expression induced by LPS stimulation (Figure 4(D,E)). Furthermore, JGP was found to inhibit the expression of cleaved Caspase-3.

Figure 4. JGP inhibits the proliferation and migration of RAW264.7 cells by inhibiting the IL-6/STAT3 signaling pathway. RAW264.7 cells were treated with JGP or LPS for 24 h. (A) The expression of TLR4 in RAW264.7 cells observed by RT-PCR. (B,C) The expression of TLR4 observed by Western blotting (B) and the quantified protein expression level of TLR4 (C). (D,E) The expression of STAT3 observed by Western blotting (D) and the quantified protein expression level of STAT3 (E). (F,G) Expression of cleave-caspase-3 and caspase-3 observed by Western blotting (F) and quantified protein expression levels of cleave-caspase-3 and caspase-3 (G). The experiment was repeated in triplicate and the results are presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

Previous research has highlighted the crucial role of caspase-3 in cell proliferation and migration. In our study, STAT3 overexpression promoted caspase-3 activation in RAW264.7 cells, while JGP effectively reduced the caspase-3 activation level in RAW264.7 cells. These findings strongly suggest that JGP hampers the proliferation and migration of RAW264.7 cells by suppressing the IL-6/STAT3 signaling pathway.

JGP regulates macrophage M1/M2 polarization

The LPS and IFN-γ receptors can induce macrophage M1 polarization, and IL-4 combined with IL-13 can induce macrophage M2 polarization. RT-qPCR results showed that LPS and IFN-γ treatments significantly up-regulated CD86 expression compared to the control. Compared to the control serum group, JGP significantly inhibited the expression of the M1 marker CD86 in RAW264.7 cells (Figure 5(A)). With the increase in JGP concentration, CD86 expression gradually decreased. In addition, JGP promoted the expression of the M2 marker Mrc1 (Figure 5(B)). These results suggest that JGP treatment inhibits macrophage M1 polarization and promotes M2 polarization.

Figure 5. JGP regulates macrophage M1/M2 polarization. (A) The expression of CD86 observed by RT-PCR. (B) Mrc1 expression was observed by RT-PCR. The experiment was repeated in triplicate and the results are presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

Discussion

Liver injury refers to chronic liver damage caused by various factors, such as viral hepatitis, alcoholic liver disease and drug-induced liver damage (Andrade et al. 2019; Treem et al. 2021). The fundamental mechanisms of liver injury include immune-mediated and chemical liver injury. The most common clinical type of liver injury is immune-mediated, which includes viral hepatitis, autoimmune liver disease and drug-induced liver injury. The pathological process underlying immune-mediated liver injury primarily involves innate immune-mediated inflammatory reactions (Zhang J et al. 2019, 2020). Kupffer cells (KCs), also known as liver macrophages, are the predominant effector cells involved in immune-mediated liver injury and constitute the largest population of macrophages within liver tissues. Macrophages near the portal vein in the liver serve as proficient phagocytic scavengers that efficiently remove degradation products from intestinal bacteria. KCs possess numerous immunosensitive receptors, including TLR and liver X receptors (Rivera et al. 2007; Endo-Umeda et al. 2018). Through these receptors, KCs can release pro-inflammatory mediators that contribute to the initiation and progression of inflammation. Consequently, macrophage-mediated immune and polarization responses represent the primary mechanisms underlying liver injury.

The traditional Chinese medicine compound JGP is an established prescription for empirically treating chronic liver injury, demonstrating its efficacy in improving liver function within chronic liver disease through clinical validation. In this study, we used RAW264.7 cells, a mouse mononuclear macrophage leukemia cell line induced by the Abelson mouse leukemia virus in BALB/c mice, as a model to examine the influence of drugs on macrophage proliferation, migration, polarization and inflammatory signaling pathways. The ultimate goal is to unravel the underlying mechanism through which JGP ameliorates immune-mediated liver injury.

Regulation of macrophage polarization plays a pivotal role in controlling inflammation. Janus kinase (JAK)-STAT, a classic cytokine pathway, is an essential signal transduction pathway in the inflammatory response and macrophage activation (Jimenez-Garcia et al. 2018; Wang, Cai, et al. 2023). TLR4 serves as the primary receptor in the LPS-triggered macrophage immune response. Upon activating TLR4, LPS can stimulate macrophages to secrete numerous inflammatory factors, which play a crucial role in the inflammatory response (Ye et al. 2019; Li et al. 2021). JGP contains various active ingredients that are associated with immune cells and inflammation. Ginseng, Astragalus, and Salvia miltiorrhiza regulate and inhibit LPS-induced inflammatory development by modulating macrophage polarization (Cheung et al. 2013; Chen et al. 2019; Wang, Ji, et al. 2023). LPS can activate downstream nuclear factor kappa B (NF-κB) and cAMP response element-binding (CREB) protein signaling through TLR4, promoting the release of inflammatory factors, such as soluble IL-1 (sIL-1), IL-6 and TNF-α by macrophages. TLR4 can also stimulate macrophages to secrete IL-1 through STAT3 (Swanson et al. 2020). Previous studies have shown that STAT3 signaling also plays a crucial role in macrophage polarization (Yin et al. 2018; Zhong et al. 2022), and STAT3 activation can promote the macrophage M2 polarization process.

In this experiment, we discovered the crucial role of STAT3 in macrophage function. STAT3 overexpression enhanced macrophage proliferation and migration while activating downstream factors such as TLR4 and caspase-3. In contrast, JGP demonstrated the ability to inhibit macrophage proliferation and migration and suppress STAT3 expression, subsequently affecting downstream protein expression. In particular, JGP showed the ability to promote macrophage M2 polarization and reduce inflammation expression. This intriguing finding suggests that JGP may induce macrophage M2 polarization through alternative signaling pathways. This area is the subject of future exploration by our team.

Macrophages can undergo phenotypic changes and alterations in function due to various factors. Activated macrophages are commonly classified as M1-like macrophages or M2-like macrophages (Wang, Ma, et al. 2021). The M1 and M2 macrophages play significant roles in inflammatory responses, with the M1 macrophages involved primarily in pro-inflammatory responses and the M2 macrophages involved mainly in anti-inflammatory responses (Liu et al. 2014). Modulating the activation state of macrophages to improve the inflammatory environment has proven to be an effective therapeutic approach to treating diseases (Wang, Xi, et al. 2021). Our research reveals that JGP inhibits M1 polarization and promotes M2 polarization of RAW264.7 cells. The findings of this study suggest that JGP can modulate macrophage M1/M2 polarization, leading to anti-inflammatory effects.

Conclusions

This study demonstrates that JGP can inhibit macrophage proliferation and migration, promoting M2 polarization. This action attenuates inflammation and confers protection against liver injury and fibrosis. Modulation of macrophage proliferation and migration by JGP may occur through the IL-6/STAT3 signaling axis. This study offers a theoretical and experimental foundation for applying JGP in clinical practice.

Authors’ contributions

KL performed the experiments, authored, reviewed the article drafts and approved the final draft. XZ and JZ conceived and designed the experiments wrote or reviewed drafts of the article, and approved the final draft. ZY and YJ analysed the data, prepared figures and/or tables and approved the final draft. FG and FZ analysed the data, prepared figures and/or tables, authored or reviewed article drafts and approved the final draft.

Disclosure statement

The authors declare no potential conflicts of interest.

Additional information

Funding

This work was supported by the Science and Technology Program of Nantong City, Jiangsu Province (MSZ20192), Scientific Research Project of Nantong Municipal Health Commission (QA2020048, MS2022102), The Medical Research Project of Jiangsu Provincial Health Commission (M2022024) and Natural Science Foundation of Nanjing University of Traditional Chinese Medicine (No. XZR2020084).