Corydalis decumbens Alleviates the Migration, Phagocytosis, and Inflammatory Response of Macrophages

Background The role of Corydalis decumbens (CD) in macrophage activation remains unclear, particularly in the Ras homolog family member A (RhoA) signaling pathway. Therefore, the present study aimed to investigate the effect of CD on the viability, proliferation, morphological changes, migration, phagocytosis, differentiation, and release of inflammatory factors and signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Methods Cell counting kit-8 and water-soluble tetrazolium salt assays were used to evaluate the viability and proliferation of RAW264.7 macrophages. A transwell assay was examined to assess cell migration. The ingestion of lumisphere assay was employed to detect the phagocytic capacity of macrophages. Phalloidin staining was performed to observe morphological changes in the macrophages. An enzyme-linked immunosorbent assay was performed to quantify inflammation-related cytokines in cell culture supernatants. Cellular immunofluorescence and western blotting were adopted to show the expression of inflammation-related factors, biomarkers of M1/M2 subset macrophages, and factors of the RhoA signaling pathway. Results We found that CD increased the viability and proliferation of RAW264.7 macrophages. CD also impaired the migration and phagocytic capacity of macrophages, induced anti-inflammatory M2 macrophage polarization, such as M2-like morphological changes, and upregulated M2 macrophage biomarkers and anti-inflammatory factors. We also observed that CD inactivated the RhoA signaling pathway. Conclusions CD mediates the activation of LPS-stimulated macrophages, alleviates the inflammatory responses of macrophages, and activates related signaling pathways induced by LPS.


Introduction
Corydalis decumbens (CD) is a traditional Chinese medicine included in the 2020 edition of the Chinese Pharmacopoeia. It possesses analgesic efects and has shown promise as a potential clinical treatment for sciatica; however, the underlying mechanism remains unclear. Recently, we reported that CD could have high efcacy as an antiinfammatory, detumescent, and analgesic agent in rat models with sciatica [1]. Furthermore, CD reduced macrophage infltration and downregulated the expression of proinfammatory cytokines in the microenvironment of damaged sciatic nerves. Based on these results, we investigated the mechanism of CD's functional regulation of macrophages.
Macrophages are important immunological cells crucial for maintaining and restoring homeostatic physiological conditions and play a major role in infammation in many neurological diseases, including sciatica [2][3][4][5]. Following peripheral nerve damage, macrophages infltrate into the injured nerve microenvironment and play key roles in initiating phagocytic activities and facilitating infammatory responses to enhance peripheral sensitization. Tis increases central sensitization and causes persistent pain. Upon polarization state, macrophages are classifed as inactivated macrophages (M0 macrophages), classically activated macrophages (M1 macrophages), and alternatively activated macrophages (M2 macrophages) [6,7]. Activated M1 macrophages may aggravate infammatory responses, and a blockage of M1 cells can alleviate neuroinfammation. In contrast, M2 macrophages are responsible for eliminating infammation and initiating tissue repair [8,9]. In this study, we chose lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and explored cellular physiological characteristics, such as cell proliferation, polarization, migration, phagocytosis, and cytokine secretion, to analyze the antiinfammatory mechanism of CD. In addition, we investigated the signaling pathways involved in macrophage activation. Existing data have illustrated that macrophage activation is closely related to the Ras homolog family member A (RhoA) signaling pathway [10][11][12]. Activated macrophages require dynamic reorganization of cytoskeleton components through RhoA regulation to exert multiple functions, such as phagocytosis, migration, and cell-to-cell interactions. A decrease in RhoA signaling pathway activity in macrophages could inhibit M1 polarization and reduce infammatory responses [13,14]. To test whether CD plays a role in the RhoA signaling pathway, which remains unclear, we investigated the expression of RhoA and its effector, Rho-associated protein kinase (ROCK), in macrophages. Overall, our aim was to explore the efects of CD on the activity of LPS-stimulated macrophages to attenuate infammatory responses and the signaling pathway involved. To establish the infammation model, 1 μg/ mL LPS (Solarbio, China) was added to the cultured cells and incubated for 24 h. CD injection (Herbi-Sky, China) was then added for 4 h. In this study, the cells were divided into four groups: control (no LPS and CD added), LPS (LPS added), LPS + CD (LPS and CD added), and CD (no LPS added).

Cell Viability Assay.
Cell viability was examined using the Cell Counting Kit-8 (CCK-8) assay (MCE, USA). Cells were frstly seeded in a 24-well plate at a density of 1 × 10 5 cells/well in DMEM/F12 medium. Furthermore, 1 μg/mL LPS was added to the LPS group and the LPS + CD group, respectively. After incubating for 24 h at 37°C, the cells were resuspended and seeded in a 96-well plate at a density of 1 × 10 4 cells/well. CD was then added to the LPS + CD and CD groups and then incubated for 4 h. Tereafter, 10 μl CCK-8 solution was added to each well, and the cells were incubated for 1 h at 37°C. Te absorbance at 450 nm was shown using a microplate reader (Tecan, Switzerland).

Cell Proliferation
Assay. Cell proliferation was detected using a water-soluble tetrazolium salt (WST) assay (Abcam, UK). Macrophages were cultured in the same conditions as above. 10 μL WST reagent was added to 0.1 mL of medium in each well of 96-well plates and incubated for 1 h at 37°C. Te absorbance at 450 nm was measured using a microplate reader.

Migration Assay.
Macrophage migration was evaluated with a transwell cell migration assay as previously described [15]. Briefy, the chambers with 8 μm pores (Corning Costar, USA) were frstly pretreated with DMEM/F12 medium. Ten, 100 μL of DMEM/F12 containing 1 × 10 5 macrophages and 1% FBS was added into the upper chamber, and the lower compartment was flled with 600 μL medium containing 10% FBS without cells. After incubation for 18 h, the transwell membrane was fxed with 4% paraformaldehyde (PFA) (Macklin, China) for 30 min. After removing the nonmigrated cells from the upper chamber with a cotton swab, the migrated cells were stained using a 0.1% crystal violet solution for 30 min. For quantifcation, 6 pictures of each membrane were photographed with an inverted microscope (Leica, Germany).

Phagocytic Capability Assay.
Te ingestion of lumisphere was used to assess phagocytosis in macrophages [16]. Macrophages were grown on 24-well chambered slides at a density of 1 × 10 5 cells/well. Tese cells were incubated for approximately 16 hours, and then 0.1 mg/mL fuorescent green microspheres (1 μm in diameter, BaseLine, China) were added to the culture medium for 3 h. Cells were then fxed with 4% PFA. Te number of lumisphere endocytosed by each cell was calculated (20 cells per group were evaluated) by fuorescent microscopy (Nikon, Japan).

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Evidence-Based Complementary and Alternative Medicine 2.8. Immunofuorescence. In this study, 1 × 10 5 cells were frstly fxed by immersion in 4% PFA for 20 min and then infltrated in 1% Triton X-100 for 1 h and incubated with 5% BSA in PBS for 30 min. Te cells were then treated with primary antibodies for at least 24 h at 4°C, followed by fuorescent-conjugated secondary antibodies for 2 h. Finally, the slides were mounted using Vectashield medium. Te stained cells were observed by fuorescent microscopy, and immunostained pictures were photographed using a highresolution digital camera.

Western
Blotting. Cells were harvested, and the proteins were extracted from collected cells using RIPA lysis bufer (CST, USA), run on 10%-12% SDS-PAGE gels, and then transferred from the gels to polyvinylidene difuoride membrane (PVDF) (Millipore, USA). Te membrane was blocked in 5% BSA for 2 h, stained with the primary antibody for 24 h at 4°C, and then blots were probed with secondary antibodies for 2 h. Enhanced chemiluminescence was visualized on the PVDF membrane, and the band density was quantifed using Image-Pro Plus 6.0.

Enzyme-Linked Immunosorbent Assay.
Te supernatants of cultured macrophages were assayed using an enzyme-linked immunosorbent assay (ELISA) (Boster, USA) array following the manufacturer's protocol to measure the expression of proinfammatory cytokines TNF-α and IL-6 and anti-infammatory factor IL-10. Te culture supernatants were collected and added to each ELISA plate. Te absorbance at 450 nm was measured by a microplate reader.
2.11. Image Analysis. For quantifcation of fuorescence intensity, the images in 3 sections per group were taken at 40x magnifcation in areas, and 6 random felds per section were considered. Te result was quantifed with Image-Pro Plus 6.0.

Statistical
Analysis. SPSS 23 software (IBM, USA) was used for the statistical analyses. Each independent experiment was performed in triplicate, and the results were presented as the mean ± standard error. Te diference between groups was analyzed by one-way analysis of variance, or a student's t-test. Te statistical signifcance was set at P < 0.05.

CD Upregulates the Viability and Proliferation of LPS-Stimulated
Macrophages. Quantitative analysis of the cultured cells was performed using CCK-8 and WST assays. We observed the efect at three diferent CD concentrations (0.25, 0.5, and 1 μL/mL), with and without LPS stimulation, and summarized the relative cell viability and proliferation after the incubation (100% for untreated cells at 24 h). Te data showed that the viability of macrophages treated with the three concentrations of CD without LPS stimulation did not change (Figure 1). Tese results suggest that CD has no cytotoxic efect on RAW264.7 macrophages. In addition, LPS-stimulated cells exhibited signifcantly lower viability and proliferation than those in the other groups. When CD was added, cell viability and proliferation increased, particularly in the 0.5 μL/mL group. As a result, 0.5 μL/mL CD was available during the experimental period.

CD Suppresses the Migration and Phagocytic Capacity of
Macrophages. In this study, we focused on the comparison of the cultured macrophages between the LPS and LPS + CD groups. Firstly, we visualized the migrated cells in the transwell lower chamber to compare the chemotactic ability of macrophages. We found that macrophage migration was signifcantly enhanced after LPS stimulation in the LPS group. Compared to the macrophages in the LPS group, the cells showed a slower migration rate in the LPS + CD group ( Figure 2). Next, we examined whether CD altered the phagocytic capability of macrophages. Macrophages were labeled with CD68 (a macrophage marker) and 0.1 mg/mL FITCconjugated lumisphere. After LPS treatment, the number of macrophages that were fuorescence-labeled by lumisphere ingestion increased, but there was a greater decrease in internalized lumisphere within each macrophage in the LPS + CD group than in the LPS group (Figure 3).

CD Induces Anti-Infammatory M2 Macrophage
Polarization. To study macrophage polarization, we observed changes in cellular morphology. Mounting evidence [16,17] suggests M1-polarized macrophages were typically fattened with lamellipodia-like protrusions, while M2polarized macrophages were elongated into spindle-like shapes. In this study, macrophage morphology was examined by FITC-phalloidin staining. We observed that the cells exhibited diferent shapes (Figure 4). In the untreated control and CD groups, the macrophages were generally round. Compared to the cultured macrophage in the LPS group, there were fewer cells that formed lamellipodia-like structures and more cells that elongated their shape in the LPS + CD group.
Additionally, M1/M2 macrophage polarization changes were compared to explain macrophage responses during infammation caused by LPS. Immunofuorescence staining showed that the M1 population biomarker, iNOS, increased in the group with LPS-stimulated macrophages but not in the LPS + CD group. Te LPS-stimulated cells had a larger population of M1 macrophages than CD-treated cells. Meanwhile, the M2 population marker, CD163, was increased in the macrophages of the LPS + CD group but not in those of the LPS group ( Figure 5). To further illustrate the macrophage subset population, western blotting was performed. Te results showed signifcant upregulation of M1 biomarkers (iNOS and CD80) and downregulation of M2 biomarkers (CD163 and CD206) ( Figure 6). Tese fndings suggest that CD treatment of macrophages may increase macrophage polarization towards the M2 phenotype.

Evidence-Based Complementary and Alternative Medicine
Furthermore, we considered the production of infammatory factors, TNF-α, IL-6, and IL-10. Expression of the proinfammatory factors, TNF-α and IL-6, enhanced signifcantly in the macrophage after LPS stimulation.
However, the expression of proinfammatory factors in macrophages in the LPS + CD group was considerably lower than that in the LPS group (Figures 7(a)-7(d)). Meanwhile, CD-induced anti-infammatory factor IL-10  expression increased signifcantly in macrophages of the LPS + CD group compared to that in the LPS group. Finally, we studied the production of infammatory cytokines using ELISA. Although not all diferences were statistically signifcant, the trend in proinfammatory or anti-infammatory factor secretion was consistent with the fuorescence results (Figures 7(e)-7(g)). Tese results suggest that the downregulation of proinfammatory factors TNF-α and IL-6 and upregulation in antiinfammatory cytokine IL-10 expression in macrophages was triggered by CD treatment in the LPS + CD group.

CD Inactivates the RhoA Signaling Pathway.
To evaluate whether the RhoA signaling pathway is responsible for mediating CD treatment of LPS-stimulated macrophages, RhoA and ROCK were detected. Compared to the LPS group, expressions of RhoA and ROCK were downregulated in the presence of CD in the LPS + CD group (Figures 8(a)-8(c)). Western blotting verifed that expressions of RhoA and ROCK were remarkably decreased in the LPS + CD group (Figures 8(d)-8(f )). Tese results led us to hypothesize that the RhoA pathway may be associated with the suppression of CD by LPS stimulation in macrophages.

Discussion
An increasing number of reports have shown that the LPSstimulated RAW264.7 macrophage model is highly adaptable to infammatory response research [18][19][20]. In vitro stimulation with LPS can drive the polarization of M1 macrophages, regardless of morphology or function [21,22]. Herein, we developed an in vitro study and found that LPS stimulation induced the upregulation of phagocytosis, migration, and expression of proinfammatory factors, as well as the downregulation of proliferation and antiinfammatory factor expression in cultured macrophages. Tese results agree with those of previous reports [23][24][25].
In this study, we found that CD increased the viability and proliferation of RAW264.7 macrophages (Figure 1) and suppressed macrophage migration and phagocytosis (Figures 2 and 3). Tese fndings reveal that CD partially infuences LPS stimulation of macrophages by alleviating macrophage migration and phagocytosis, the two important biological processes during infammatory responses [26,27].
As is well known, macrophages can trigger infammatory responses and play a crucial role in altering proinfammatory or anti-infammatory microenvironments [28][29][30]. Phenotypic switching plays a key role in eliminating infammation. In this study, we observed that CD treatment changed the shape of macrophages, elongating them to form M2-like Evidence-Based Complementary and Alternative Medicine 5 macrophages ( Figure 4). Generally, the M1 subtype macrophage produces abundant proinfammatory cytokines that hinder tissue repair. Compared to the proinfammatory M1 macrophages, the M2 type is capable of secreting antiinfammatory factors and reducing infammatory responses [31,32]. Our study found that CD enhanced the expression of M2 biomarkers in LPS-stimulated macrophages, such as CD163 and CD206 (Figures 5 and 6). Tus, we postulated that CD efects on LPS-stimulated macrophages do not induce strong infammation but are helpful for the recovery of injured cells through M2 polarization. Furthermore, the upregulated secretion of the antiinfammatory factor, IL-10, and downregulated production of proinfammatory cytokines, TNF-α and IL-6, in the culture supernatants strongly implied that CD-induced M2 phenotype polarization, forcefully shifted the macrophages from M1 to M2 subset, and mediated the antiinfammatory efects via the regulation of cytokines (Figure 7).
Te RhoA signaling pathway is closely related to the activation of macrophage development and infammatory responses. Regulation of the cytoskeleton through the RhoA pathway has been demonstrated to alter the shape and function of macrophages [33,34]. Choi et al. [13] found that Rhosin, a RhoA inhibitor, can prevent RhoA interaction with its GEFs and decrease M1 macrophage polarization and infammatory responses by modulating dynamic changes in the actin cytoskeleton. Inactivation of RhoA GTPase can initiate multiple consequences on host cells, such as suppression of cell migration or cell death [35,36]. Our study found that RhoA and ROCK expression were downregulated upon CD treatment (Figure 8). Tis result suggests that RhoA control may afect macrophage subset polarization and function by causing morphological and functional changes in macrophages, although the detailed mechanisms are yet to be elucidated.
Taken together, the novel fndings of this study were as follows: CD (1) increased the proliferation and viability of   macrophages, (2) polarized macrophages towards M2 cells, (3) inhibited migration and phagocytic capacity, (4) contributed to M2 chemokine marker expression in macrophages, and (5) decreased pro-infammatory factor expression while increasing anti-infammatory factor expression.

Conclusions
Our fndings highlight that CD can regulate the activation of LPS-stimulated macrophages and exert anti-infammatory efects. In addition, the RhoA signaling pathway is vital in CD treatment, and more intensive studies are needed for further research.

Data Availability
Te data presented in current study are available within this article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.