Effect of Chuanzhifang component (ZGC) on macrophage inflammatory injury based on whole gene expression profile

JIANG Jie, AN Wan-li, YANG Zhi-qian, CHENG Wen-hui, YANG Hong

Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China

Keywords:

ABSTRACT Objective: The effect of Chuanzhi Fang (ZGC) on the whole genome expression profile of RAW264.7 cells activated by lipopolysaccharide (LPS) was analyzed, and to explore the possible mechanism of action and core target of this formula on macrophage inflammatory injury at the overall level.Methods: A model of LPS-induced inflammation in RAW264.7 cells was constructed, and the effect of ZGC intervention on the genome-wide expression of inflammatory macrophages 3was examined by gene microarray technology, GO/KEGG enrichment analysis was performed for significantly differentially expressed genes among each group.Results: The results of genome-wide expression profiling microarray analysis showed that the ZGC intervention group upregulated the expression of 5 genes including C4bp and inhibited the expression of 22 genes including Mgat3, Psma6, and Siglecg relative to the LPS model group.KEGG signaling pathway analysis results showed that ZGC mainly acted through cytokine receptor interaction and the C-type lectin receptor signaling pathway.Conclusion: ZGC can interfere with the abnormal expression of 27 genes in inflammatory macrophages, and the related genes may exert corresponding anti-inflammatory effects by affecting cytokine receptor interactions, C-type lectin receptor signaling pathway, and TLR4/NF-κB signaling pathway.

1.Introduction

Inflammatory and immune cells play an important role in the development of a variety of systemic diseases, especially macrophages in the intrinsic immune cells, with phagocytosis,capable of recognizing and clearing a variety of pathogens,secreting a variety of inflammatory cytokines after stimulated classical activation, and initiating the activation of a series of inflammatory signaling pathways, which play an important role in defense against pathogenic infections and tissue inflammation in the organism.Macrophages are widely distributed and can survive up to several months, and at the same time have functional plasticity and heterogeneity, which not only participate in the regulation of inflammatory injury related to infectious diseases such as sepsis and acute lung injury, but also their local inflammatory infiltration is closely related to some non-infectious diseases, such as cerebral ischemia/reperfusion injury, atherosclerosis, diabetes mellitus, and so on, and is a key participant in aggravating the process of the disease and tissue injury.Therefore, macrophages have become a potential therapeutic target for inflammatory-responsive diseases,and the development of drug research and clinical therapeutic strategies centered on the modulation of macrophage inflammation is of great importance.

Based on the antagonism of macrophage inflammation and the development of traditional Chinese medicine component Chuan gardenia formula (ZGC) anti-inflammatory effect is clear, the group of the previous series of studies[1] showed that, for the LPS-induced inflammatory activation of macrophage, ZGC can be as many as 16 kinds of inflammatory factors have a significant inhibitory effect,such as IL-6, IL-1 , IFN-γ, etc., the overall animal experiments have also confirmed that it has a protective effect on cerebral ischemia-reperfusion injury[1] and septic acute lung injury, which is mainly characterized by the pathology of the inflammatory cascade response.The overall animal experiments also confirmed its protective effect on cerebral ischemia/reperfusion injury[1] and acute lung injury in sepsis, which are mainly characterized by the pathological cascade of inflammation, and proved the feasibility and importance of developing drugs with the therapeutic target of regulating macrophage inflammation.However, the specific molecular mechanism of ZGC’s anti-macrophage inflammation is still unclear, for this reason, the present study used highthroughput gene chip assay as the core technology to explore ZGC’s role in regulating the genome-wide expression of inflammatory macrophages and its core targets, so as to provide a scientific explanation of its mechanism of action, and to lay the experimental foundation for the further in-depth study of anti-macrophage inflammation traditional Chinese medicine.

2.Information and methodology

2.1 Experimental materials

Mouse RAW264.7 (Xiehe Cell Bank).High glucose DMEM,FBS(Gibco); LPS (Sigma, L4391), DMSO (Sigma); Gardenia glycosides, Puerarin, Tetramethylpyrazine(Shanghai Tongtian Biotechnology Co.); CCK-8 (Dojindo); Mouse IL-6 ELISA kit(MultiSciences Biotech Co); Gel Red (Biotium); RNA standard product (Thermofisher); Nucleic acid molecular weight marker(SMOBIO); Ambion™ Amino Allyl MessageAmp™ II aRNA Amplification Kit (Thermofisher); Agilent RNA 6000 Nano Kit (Agilent); Labeling Reagent and Hybridization Kit, Mouse OneArray® (Phalanx Biotech).2100Bioanalyzer;Microarray scanner(Agilent).

2.2 Experimental Methods

2.2.1 Drug preparation

Preparation of LPS: Weigh appropriate amount of LPS powder, use high sugar DMEM to formulate into 4, 2, 1 and 0.5 μg/mL solution,and store in separate packages.

Preparation of ZGC: Dissolve gardenia glycoside, chuanxiongzine and geranylgeranyl powder in high sugar DMEM, prepare ZGC solution at 800, 600, 400 and 200 μg/mL, dissolve with ultrasonication and filter with 0.22 μm filter membrane.

2.2.2 cell culture

RAW264.7 cells were cultured at 37 ℃, 5% CO2, 10% FBSDMEM to avoid stimulation and passaged every other day.

2.2.3Modeling of macrophage inflammation

RAW264.7 cells were inoculated in 6-well plates and divided into four groups: LPS ultra-high (4 μg/mL), high (2 μg/mL), medium(1 μg/mL) and LPS low-dose model group (0.5 μg/mL), and then incubated in a constant temperature incubator at 37 ℃ with 5% CO2for 24 h, and then converted to the corresponding concentration of LPS solution.IL-6 concentration was measured by ELISA.

2.2.4Toxic effects of LPS on RAW264.7 cells detected by CCK-8 assay

Cells were divided into LPS model group (1 μg/mL) and Control group, and incubated at 37 ℃ with 5% CO2for 24 h.According to the experimental groups, the supernatant was replaced with 1 μg/ml LPS solution and high sugar DMEM, respectively, and after 20 h, the supernatant was replaced with high sugar DMEM containing 10% CCK-8, and the cells were incubated at 37 ℃ and protected from light for 0.5 H.After incubation, the OD values were measured and the cell survival and inhibition rates were calculated.calculate the cell survival and inhibition rate.

2.2.5 Experimental Dosage Examination of ZGC Cells

According to 800 μg/mL, 600 μg/mL, 400 μg/mL and 200 μg/mL,the cells were divided into four experimental groups of ultra-highdose/high-dose/medium-dose/low-dose administration and Control group, and the operation was the same as in step 2.4.After 24 h, the original supernatant was discarded, 10% CCK-8 solution was added,and the cells were incubated for 0.5 h at 37 ℃ away from the light for the OD value to be detected.After incubation at 37 ℃ for 0.5 h,the OD value was measured, and the cell survival and inhibition rates were calculated.

2.2.6ZGC anti-LPS induced RAW264.7 inflammatory injury assay

RAW264.7 cells were inoculated into 6-well plates and divided into Control group, LPS model group (1 μg/mL), and ZGC administration group (400 μg/mL), after 24 h of incubation, the supernatants of the cells in the normal control group and the LPS model group were replaced with a new high-sugar DMEM solution,and those in the ZGC group were replaced with the ZGC solution with the final concentration of 400 μg/mL, and after incubation for 4H.After incubation for 4 h, the LPS and ZGC groups were added with a final concentration of 1 μg/mL of LPS solution, and the Control group was added with the same volume of DMEM 20H later, the supernatants were collected, and the concentration of IL-6 was detected by ELISA, and the cells in each group were lysed by Trizol and then frozen at -80 ℃ for spare use.

2.2.7RNA extraction quality control, microarray detection and analysis

Total RNA was extracted from cells, followed by rigorous quality control testing of the RNA samples.Subsequently, the samples were hybridized onto gene chips and chip signals were scanned.The obtained data was then subjected to analysis for differential gene expression.To further enhance the scientific rigor, online bioinformatics tools (https://www.bioinformatics.com.cn.) were used to perform cluster analysis and functional enrichment analysis.

2.3 Statistical Analysis

The data was processed and analyzed using SPSS 20.0 software,with the results presented as (±s).Intergroup differences in the data were examined through one-way analysis of variance (ANOVA).

3.Experimental Results

3.1 Results of LPS dosage screening

The ELISA results demonstrated that, in comparison to the Control group, all LPS groups exhibited a significant capacity to induce an elevation in IL-6 secretion by cells.Moreover, there was a positive correlation between the concentration of LPS stimulation and the expression level of IL-6.The detailed outcomes are presented in Table 1.

Tab 1 Each dose of LPS stimulated RAW264.7 cells to secrete different concentrations of IL-6(n=3, ±s)

Tab 1 Each dose of LPS stimulated RAW264.7 cells to secrete different concentrations of IL-6(n=3, ±s)

Group Concentration of LPS (μg/ml) Concentration of IL-6 (pg/ml) P Control group 0 0 -LPS low-dose model group 0.5 8 185.52±947.02 ＜0.001 LPS medium-dose model group 1 14 603.57±1 179.27 ＜0.001 LPS high-dose model group 2 18 390.66±631.67 ＜0.001 LPS ultra-high-dose model group 4 19 638.61±1 202.94 ＜0.001

3.2 Results of the LPS cytotoxicity experiment

The cytotoxicity experiment results revealed that the stimulation of RAW264.7 cells with 1 μg/mL LPS for a duration of 20 h did not exhibit any impact on cell proliferation.Considering previous experimental investigations, the concentration of 1μg/ml was chosen as the optimal condition to induce cellular inflammatory response using LPS.The corresponding outcomes are presented in Table 2.

Tab 2 Results of LPS cytotoxicity experiments (n=4)

3.3 Results of ZGC cytotoxicity experiment

The CCK8 results demonstrated a positive correlation between the concentration of ZGC and the inhibition rate of cell proliferation.Drawing upon previous experimental research experience and findings, ZGC with a concentration of 400 μg/mL was selected 400 μg/mL as an optimal intervention condition for further investigation.The corresponding outcomes are presented in Table 3.

Tab 3 Cell survival study of ZGC action on RAW264.7 cells at 24 h (n=4)

3.4 Confirmation of the efficacy of ZGC in attenuating inflammation in RAW264.7 cells

As a classical pro-inflammatory factor in cellular inflammation,a significant increase in IL-6 secretion was observed in the LPS group, indicating successful establishment of the cellular inflammation model.In comparison to the LPS group, there was a noteworthy reduction in IL-6 levels observed in the ZGC group,thereby confirming its effective suppression of IL-6 release levels in RAW264.7 inflammatory models.These findings align with previous experimental research and provide evidence for the definite anti-inflammatory effects exhibited by ZGC.Detailed experimental results are presented in Table 4.

Tab 4 Effect of ZGC on the concentration of IL-6 secreted by RAW264.7 inflammation model(n=3, ±s)

Tab 4 Effect of ZGC on the concentration of IL-6 secreted by RAW264.7 inflammation model(n=3, ±s)

Note: Comparing LPS model group to Control group : #P＜0.05, ##P＜0.01.Comparing ZGC intervention group to LPS model group : *P＜0.05, **P＜0.01.

Group Concentration of IL-6 (pg/mL) P Control group (C) 0 -LPS model group (M) 14 663.23±300.25## ＜0.001 ZGC intervention group (MZGC) 12 552.11±1 002.45* 0.025

3.5 Extraction and quality assessment of RNA

The provided RNA was quantitatively analyzed using absorbance spectroscopy, resulting in satisfactory quality control outcomes.The RNA demonstrated high purity without any contamination and exhibited excellent integrity.Experimental findings are presented in Table 5.

3.6 Results of whole-genome expression profiling microarray for mice

3.6.1 Investigation into Differential Gene Expression Patterns

Fig 1 Histogram of fold difference

Fig 2 Volcano map

Gene chip scanning images were obtained by scanning gene chips,and the signals at different sites on the chip were analyzed to obtain normalized data for differentially expressed genes.By applying the criteria of |Fold change| 0.585 and P ＜ 0.05, significantly differentially expressed genes were identified.In comparison with the control group, 136 upregulated genes and 25 downregulated genes were observed in the LPS group; while in comparison with the LPS group, there were 53 downregulated genes and 8 upregulated genes in the ZGC group.Detailed experimental results are presented in Figure 1-2 and Table 6.By further selecting more significantly differentially expressed genes using a |Fold change| 1 and P ＜0.05 threshold, 22 downregulated genes and 5 upregulated genes were identified in the ZGC group compared to the model group as shown in Table 7.

Tab 5 Quantitative results of RNA absorption spectroscopy analysis

Tab 6 Common differentially expressed genes

3.6.2Cluster analysis

Hierarchical clustering analysis was employed to identify clusters among the top 30 genes exhibiting the most significant differences in gene chip scanning results.A heatmap was generated to visualize the expression levels and correlations of these differentially expressed genes.The experimental findings are presented in Figure 3.

Fig 3 Hierarchical clustering map of differential gene expression

3.6.3 Functional enrichment analysis

Differential gene enrichment analysis between the ZGC group and the LPS model group revealed that ZGC primarily exerts anti-inflammatory effects through leukocyte-mediated immune regulation, regulation of prostaglandin secretion, synaptic membrane function, sialic acid binding, and immune receptor activity.KEGG enrichment analysis of differentially expressed genes in the ZGC group demonstrated that ZGC predominantly intervenes in macrophage inflammation via pathways such as cytokine receptor interaction and C-type lectin receptor signaling.The results of this analysis are presented in Figure 4-5.

Tab 7 MZGC group vs M group differentially expressed genes

Fig 4 MZGC group vs M group GO enrichment map

Fig 5 MZGC group vs M group KEGG enrichment map

4.Discussion

Targeting inflammatory macrophages holds great promise in drug discovery research and clinical treatment of inflammatory diseases.Monocytes in the bloodstream serve as precursor cells that migrate across vascular endothelium to damaged tissues, where they differentiate into macrophages.These macrophages form two distinct subgroups, M1 and M2, exhibiting different functional characteristics depending on various stimuli.Upon pathogenic stimulation, M1 macrophages undergo classical activation and release cytokines such as TNF-α/IL-1, inducible nitric oxide synthase (iNOS), and Th1 chemokines to eliminate pathogens.They also enhance intracellular production of ROS/NOS, playing a crucial role in initiating inflammatory responses.These actions promote inflammation and exacerbate tissue damage.Conversely, alternatively activated M2 macrophages produce abundant anti-inflammatory cytokines like IL-10, Th2 chemokines, C-type lectins (CTLs), etc., facilitating debris clearance and promoting angiogenesis - both essential for resolving inflammation and tissue repair[2-4].

LPS have been shown to promote the differentiation of macrophages into M1 phenotype, thereby inducing inflammatory responses[5].This phenomenon is closely associated with Toll-like receptor 4 (TLR4), which recognizes pathogen-associated molecular patterns and mediates immune-inflammatory reactions.As the classical ligand for TLR4, LPS binds to macrophage surface TLR4,triggering classical polarization and subsequent release of a plethora of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α.Additionally, it activates signaling pathways including NF-κB,MAPK, JAK/STAT, initiating cascades of inflammatory reactions that exacerbate disease progression and tissue damage[6].

Our research group has developed a traditional Chinese medicine formula called ZGC, which is based on the modulation of macrophage inflammation.It exhibits a pronounced antiinflammatory effect and comprises three constituents: gardenoside,puerarin, and ligustilide.ZGC significantly suppresses the expression of various inflammatory cytokines in macrophages such as IL-6, IL-1α, and IFN-γ[1].Differential gene analysis using gene chips revealed that administration of ZGC resulted in upregulation of 5 genes while downregulating 22 genes, leading to a total of 27 differentially expressed genes.GO enrichment analysis unveiled its involvement in leukocyte-mediated immune regulation, regulation of prostaglandin secretion, synaptic membrane function, sialic acid binding, and immune receptor activity.KEGG enrichment analysis demonstrated that ZGC primarily intervenes in macrophage inflammation through pathways associated with cytokine receptor interaction and C-type lectin receptor signaling.

Among them, the gene expression related to immune inflammation is significantly altered.Compared with the LPS model group,the ZGC group exhibits a significant upregulation in C4bp gene expression.The C4bp protein possesses the ability to inhibit complement system activation.The activation of the complement system can facilitate extensive neutrophil aggregation, leading to their adherence onto endothelial cells within blood vessels and subsequently enhancing vascular permeability.This process enables diverse immune cells to infiltrate the site of injury effectively.Nevertheless, if complement system activation occurs during the advanced stage of inflammation, it may give rise to excessive inflammatory responses and autoimmune reactions.M Kishiwada[7]demonstrated through rat experiments that LPS reduces the expression of both chains (C4BP α and C4BP β) of C4b-binding protein via NF-κB and MEK/ERK pathways, thus promoting inflammation development[7].Other studies have indicated[8] that when present on cell surfaces, C4bp plays a crucial role in preventing deposition of inflammatory complexes in tissues and maintaining self-antigen tolerance, providing protection against inflammation and autoimmunity.Research by Klaudia Kulak[9] has shown that C4bp significantly inhibits macrophage IL-1β secretion induced by pancreatic islet inflammatory cell infiltration in a dose-dependent manner during non-infectious diseases.By preventing loss of lysosomal integrity required for NLRP3 inflammasome activation in macrophages, C4BP suppresses inflammation effectively.Furthermore, experimental evidence confirms[10] that C4BP effectively prevents ectopic lymphoid structure neogenesis in elderly lupus nephritis mice by downregulating systemic and local CXCL13 levels, demonstrating its anti-inflammatory and immunoregulatory activities.Therefore, upregulation of C4bp expression can inhibit the activation of the complement system, thereby attenuating neutrophil aggregation, suppressing inflammatory reactions, exerting immunomodulatory activity, and mitigating tissue damage.In previous research conducted by our team, it has been discovered that ZGC has the potential to downregulate the secretion levels of chemokines MCP-1 and Eotaxin in an inflammation model, possibly achieved through upregulating C4bp expression.

The expression of Mgat3, which is upregulated in the model group compared to the control group, is downregulated in the ZGC group compared to the model group.Mgat3 participates in glycoprotein oligosaccharide biosynthesis and studies have suggested its close association with eosinophilic inflammation[11].Furthermore, it can catalyze diacylglycerol production and induce protein kinase C(PKC) activation[12].Different subtypes of PKC[13] play crucial roles in inflammatory responses by regulating T and B lymphocytes as well as NF-κB, MAPK, and other signaling pathways.Mutations in PKC during ischemic stroke can lead to neurodegeneration and impaired cognitive abilities[14].Conversely, iron-mediated inflammation can activate the PKC pathway through glutamate activity while promoting reactive oxygen species release[15].Inhibiting Toll-like receptor activation caused by PKC has been shown to alleviate neural inflammatory damage according to studies[16].By inhibiting Mgat3, it may be possible to mitigate brain tissue inflammatory damage caused by a series of reactions following PKC activation.Preliminary experimental results indicate that ZGC can inhibit various inflammatory factors such as IL-6 and IL-1β released after lipopolysaccharide binds to macrophage surface TLR.Considering comprehensive chip results, it suggests that ZGC’s alleviation of cerebral ischemia-reperfusion injuryinduced inflammation may be associated with downregulation of the Mgat3 gene.

Siglec-g, a CD33-related receptor encoding sialic acid-binding immunoglobulin-like lectin-G, exhibits downregulation in the ZGC group compared to the model group.It shares a high homology with CD33 in its extracellular domain and plays a crucial role in immune regulation.Siglec-g is expressed in immune cells such as B-1a cells and macrophages[17].The proto-oncogene tyrosine-protein kinase Src can inhibit the inflammatory process in sepsis, and promote the release of anti-inflammatory cytokine IL-10 in colitis mouse models[18].Siglec-g can suppress Src activation to enhance TLR4-induced release of pro-inflammatory cytokines while inhibiting the secretion of anti-inflammatory factor IL-10.Deficiency of Siglec-g reduces infiltration of inflammatory cells and inflammation within adipose tissue[19].Another study has demonstrated that lack of Siglecg inhibits TLR4-triggered secretion of pro-inflammatory cytokines, increases production of IL-10 both in vivo and in vitro,and improves survival rate in septic mice[20].The intervention effect of ZGC in the inflammation induced by macrophage TLR4 activation through LPS may be closely associated with the downregulation of Siglecg gene expression, thereby impeding the C-type lectin receptor signaling pathway and inhibiting sialic acid binding.

Psma6, a constituent of the proteasome 6 subunit, is downregulated in the ZGC group compared to the model group.It plays a crucial role in ischemic cerebrovascular disease as inflammatory reactions caused by ischemic brain injury hinder proteasomal degradation of harmful proteins leading to accumulation of ubiquitinated substrates and consequent damage or death of brain cells[21].Moreover, proteasomes can activate NF-κB signaling pathway[22]by degrading IκB inhibitors resulting in activation of NF-κB and release of downstream inflammatory factors from cells[23].Studies have demonstrated that proteasome inhibitors can alleviate brain injury in rat MCAO models through inhibiting inflammatory rectiona[24].Therefore, it is suggested that ZGC can attenuate macrophage inflammation by suppressing the activation of the NF-κB signaling pathway through downregulation of Psma6 expression, thereby mitigating inflammatory damage.

In summary, ZGC exhibits the potential to inhibit complement system activation and mitigate the inflammatory enrichment of neutrophils and eosinophils by upregulating C4bp and downregulating Mgat3.Moreover, it may suppress TLR4/NF-κB pathway activation by downregulating Siglecg and Psma6, thereby attenuating the secretion of pro-inflammatory cytokines triggered by TLR4 while enhancing IL-10 release.This facilitates macrophage polarization towards an M2 state, thereby ameliorating macrophage inflammation.Consequently, ZGC serves as a promising therapeutic target against the cascade reaction of inflammation to provide protection against cerebral ischemia-reperfusion injury[1] and sepsis-induced acute lung injury.

5.Conclusion

In this study, a macrophage inflammation model and gene chip technology were employed to investigate the impact of ZGC on global gene expression in LPS-induced inflammatory macrophages.The results demonstrated that ZGC can modulate the aberrant expression of 27 genes, including C4bp, Mgat3, Psma6, Siglecg,in inflammatory macrophages.The significantly differentially expressed genes primarily participate in leukocyte-mediated immune regulation, regulation of prostaglandin secretion, synaptic membrane function, sialic acid binding and immune receptor activity and influence cell cytokine receptor interactions, CTL signaling pathways and TLR4/NF-κB signaling pathways to exert antiinflammatory effects - which may serve as crucial targets for ZGC to counteract macrophage-induced inflammatory damage.This study holds significant exploratory significance and provides a novel direction for further experimental elucidation.

6.Declaration of Conflict of Interest and Explanation of Contributions

The present article is entirely original, devoid of any instances of plagiarism or improper citation.All authors assert that there are no conflicts of interest.

The corresponding author, Hong Yang, is responsible for project funding and oversees the design and writing of the article.The first author, Jie Jiang, handles data processing, chart creation, manuscript editing, and submission.Author Wanli An actively participates in experimental operations and contributes to data processing.Author Zhiqian Yang plays a significant role in data collection and document organization.Author Wenhui Cheng is involved in statistical analysis as well as manuscript revision.