Synergistic Regulation of Microglia Gene Expression by Natural Molecules in Herbal Medicine

The activated microglia contribute to stroke-induced neuroinflammation by upregulating the expression of a pleura of genes that are characterized as either proinflammatory or anti-inflammatory. The natural products alantolactone (Ala) and dehydrodiisoeugenol (Deh) found in Inula helenium L. and Myristica fragrans Houtt., respectively, are regularly used in traditional herb medicine, which play anti-inflammatory and antioxidant roles via regulation of canonical pathways such as nuclear factor kappa B (NF-κB) in microglia and microphages. To illustrate the full spectra of gene expression alteration in microglia treated with Ala, Deh, and the mixture of Ala and Deh (denoted as Mix), we performed RNA-seq analysis of total RNA extracted from lipopolysaccharide- (LPS-) treated microglia subsequently exposed to Ala, Deh, and Mix. While both chemicals regulated the gene expression that facilitates an anti-inflammatory polarization, the mixture exerted some distinctive synergic regulatory effect, which differed from either of the chemicals alone. Our data provide important evidence for further research on the therapeutic mechanism of traditional medicine including Eerdun Wurile (EW).

In addition, it has been found that free radical damage (reactive oxygen/nitrogen species, ROS/RNS) also plays a key role in diseases such as stroke, brain traumatic injury, and Alzheimer's disease (AD). ROS/RNS can lead to DNA damage, lipid peroxidation, and protein dysfunction, resulting in cell damage and death [12,13]. When ischemic injury occurs in the brain, it will lead to neuroinflammation, and immune cells produce a large amount of ROS, which aggravates oxidative stress injury. ROS can also activate these inflammatory cells. For example, ROS activates microglia, neutrophils, and macrophages through the NF-κB pathway [12]. erefore, antioxidants are being studied as neuroprotective therapeutic agents for brain injury. e therapeutic effect of herbal medicine such as traditional Chinese medicine (TCM) originates from the synergic power of multiple ingredients. For example, the active chemical ligustrazine in Bizheng-Tang, an eightcomponent TCM, in combination with leflunomide can alter the gene expression pattern in rheumatoid arthritis (RA) and overcome the low response of RA treatment to leflunomide alone. Previously, we fractionated and analyzed the active chemicals in the traditional Mongolian medicine Eerdun Wurile (EW) and discovered several immunomodulatory small molecules such as alantolactone, dehydrodiisoeugenol, myristicin, and costunolide that suppress proinflammatory polarization of microglia and might contribute to the overall neuroprotective activity of EW [14,15]. Alantolactone (Ala) is one of the key bioactive molecules discovered in Inula helenium L., which is used as an important ingredient of EW. Ala exhibits antineuroinflammatory effects through suppression of NF-κB and MAPK pathways [16,17]. Similarly, dehydrodiisoeugenol (Deh), the main active component of Myristica fragrans Houtt., another key material of EW, has been reported to inhibit the expression of NF-κB and other genes involved in inflammation in macrophages [18].
In the middle cerebral artery occlusion and reperfusion (MCAO/R) rat model, Ala could inhibit the expression of proinflammatory factors via suppression of NF-κB and MAPK signaling pathways [17] and had neuroprotective effect in traumatic brain injured rats [16]. In cigarette smoke-induced human bronchial epithelial cells, Ala inhibited inflammation, apoptosis, and oxidative stress by activating Nrf2/HO-1 and inhibiting the NF-κB pathway [19]. In RAW264.7 cells, Deh inhibits LPS-stimulated NF-κB activation and Cox2 expression [18]. Deh showed high hydroxyl radical scavenging activity and DPPH radical scavenging activity [20].
We hypothesize that the neuroprotective effect of EW results from the synergic effect of active chemicals including Ala and Deh. To assess the combinatory effect of Ala and Deh on microglia polarization, in this report, we investigated the impact of each single molecule as well as the mixture on the gene expression pattern of BV2 cells, focusing on the inflammation pathways. We used RNA-seq technology to confirm the synergistic regulation of gene expression in LPSstimulated microglia by natural products, Ala and Deh. Significant differential expression was discovered in the synergistic effect of the two bioactive molecules, including anti-inflammatory and antioxidant effects, which may provide the neuroprotective mechanism of EW.

RNA Isolation and Library Construction for Illumina
Sequencing. BV2 cells were cultured in a 24-well plate at 5 × 10 4 cells/well and were pretreated with 21.5 μM Ala, 1.0 μM Deh, and the mixture of 21.5 μM Ala and 1.0 μM Deh (denoted as "Mix") for 12 h. en, the cells were stimulated with LPS (100 ng/mL) in the presence or absence of Ala (or Deh or Mix). After 6 h of LPS treatment, total RNA was extracted from BV2 cells using TRIzol Reagent according to the manufacturer's specifications. e high purity (28S/ 18S ≥ 2.0) and high integrity (RIN >8.0) of RNA were evaluated using the Agilent 2100 bioanalyzer. e extracted total RNA was stored at −80°C and subsequently used for RNA-seq and RT-qPCR analyses. e total RNA was extracted, and mRNA of BV2 cells was purified by using the oligo(dT)-attached magnetic beads. Adding the fragmentation reagent, the mRNA was interrupted to small pieces, then the first strand cDNA was synthesized by random hexamer-primer reverse transcription, and then the second-strand cDNA was synthesized. e double-stranded cDNA was subjected to end repair and adenylated the 3′ ends. Finally, adaptors were ligated to the cDNA fragments. e required fragments were purified by AMPure XP beads (AGENCOURT) and enriched by PCR amplification.
e sample library was validated on the Agilent Technologies 2100 bioanalyzer.
2.6. Differential Expression Analysis. Analysis of two groups' differential expression gene was performed using DESeq2. e expression level of the transcript was calculated in fragments per kilobase million (FPKM). Genes with FDR ≤0.05 found by DESeq2 were assigned as differentially expressed. For an optimal comparison of the results, cluster analysis was performed on the differential genes of NT vs LPS, LPS vs Ala, LPS vs Deh, and LPS vs Mix.

GO and KEGG Enrichment Analysis of Differentially
Expressed Genes. In order to systematically analyze the DEGs function of our data, we used Phyper (https://en. wikipedia.org/wiki/Hypergeometric_distribution) to perform Gene Ontology (GO) (http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp/) enrichment analysis of annotated DEGs based on the hypergeometric test. e FDR ≤ 0.05 was regarded as the significant level of terms and pathways.

Statistical Analysis.
e data were analyzed using GraphPad Prism 7.0. All the results were expressed as mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparisons test. P < 0.05 was considered statistical significance.

Cell Viability.
e plant origin and structure of alantolactone (Ala) and dehydrodiisoeugenol (Deh) are shown in Figure 1. To obtain the optimal concentration of the compounds for further in vitro assay, the MTS assay was performed in this experiment. e results showed that Ala and Deh did not impact the BV2 cells (mouse microglia cell line) viability at concentrations 21.5 μM and 1.0 μM, respectively (P < 0.05) ( Figure 1). erefore, the concentrations of 21.5 μM Ala and 1.0 μM Deh were used for the following experiment.

Illumina Sequencing and Aligning to the Reference
Genome. In this study, RNA-seq was used to analyze variations in gene expression of the BV2 cells transcriptome. We sequenced 10 cDNA libraries (NT, LPS, Ala, Deh, and Mix) using Illumina (paired-end) sequencing technology and obtained 47.15 million raw reads on average. After removal of low-quality reads (quality scores <15), adaptor sequences, and ambiguous reads, 90.89% of the clean reads were aligned to the reference genome using Bowtie2. 44,887,103 (NT), 45,119,346 (LPS), 44,717,416 (Ala), 44,972,023 (Deh), and 44,763,779 (Mix) clean reads were retained for further mapping and differential expression analysis. e summary of sequencing quality and mapping is shown in Table 1.

Differential Expressed Genes (DEGs) after Ala, Deh, and
Mix Treatment. A total of 15857 genes were detected in cultured microglia. 13,703 (NT), 13,709 (LPS), 13,809 (Ala), 13,997 (Deh), and 14,058 (Mix) genes were detected in the samples, and the standard of screening differential expressed genes (DEGs) was generally FDR ≤0.05. e numbers of upregulated and downregulated genes between samples are summarized in Figure 2   To obtain gene expression difference between samples obtained from cells treated with Ala, Deh, or Mix, the transcripts that attached a condition |log 2 FC| ≥1 were further analyzed. As a result (Figure 3(a)), the lowest number of DEGs was found in LPS vs Ala (Ala group), of which 157 upregulated and 221 downregulated. Meanwhile, 373 upregulated and 584 downregulated DEGs were found in LPS vs Deh (Deh group); and the largest number of DEGs was found in LPS vs Mix (Mix group), with 754 upregulated and 964 downregulated. In contrast, downregulated genes are higher than upregulated genes. ere were 79, 72, and 789 expressed genes in the Ala group, Deh group, and Mix group, respectively (Figure 3(b)). Only 4 DEGs coexpressed between the Ala group and Deh group; 48 DEGs coexpressed between the Ala group and Mix group; and 634 DEGs coexpressed between the Deh group and Mix group. A total of 247 significantly DEGs were shared by the Ala group, Deh group, and Mix group. Results suggested that the number of DEGs between the Deh group and Mix group was larger than that between the Ala group and Mix group. e clustering heatmap exhibited the 247 DEGs (Figure 3(c)). In comparison, the number of upregulated genes by these compounds was lower than that of downregulated genes.
We selected some up-and downregulated genes after LPS treatment with or without Ala (or Deh or Mix) based on |log2FC| ≥1, FDR ≤0.05, and FPKM ≥100 and have listed them in Table 2. Stimulation of BV2 cells with LPS released cytokines such as interleukins (Il1β), colony stimulating factor (Csf3), chemokines (Ccl2, Ccl7, Cxcl10), tumor necrosis factor (Tnfα ), and iNOS enzymes (Nos2), causing inflammation. After being treated with Ala or Deh or Mix, cells were downregulated the genes encoding cytokines and iNOS enzymes. For example, RNA-seq analysis revealed that the fold change (downregulated) of Il1β expression in Ala-, Deh-, and Mix-treated groups compared with the LPS group was −9.04, −69.67, and −867.61, respectively. Mix is the mixture of same concentration of Ala and Deh, but the effect of Mix group was more downregulated than the sum of them. Similarly, the fold change of Ccl7, Ccl2, Tnfα, and Nos2 expressions in the Mix group was more downregulated than that of the Ala and Deh group. All of Ala, Deh, and Mix upregulated 13 genes, such as Ddit3, Gclm, Trib3, and Srxn1. Among them, the fold change of upregulated genes in Mix was almost the sum of the fold change of upregulated genes   Evidence-Based Complementary and Alternative Medicine in Ala and Deh. And either Ala or Deh upregulated genes are also listed in Table 2. e heatmap of these genes is presented in Figure S1. e top 20 enrichment of GO terms in the Ala group, Deh group, and Mix group were involved in similar biological process and molecular function, but some were different (Figures 4(d)-4(f )). In those top 20 GO terms, all groups were involved in GO_BP including immune system process (GO:0002376), innate immune response (GO: 0045087), and inflammatory response (GO:0006954) and GO_MF including protein binding (GO:0005515), nucleotide binding (GO:0000166), and identical protein
e Ala, Deh, and Mix showed significant downregulation of all of these DEGs expression (Figures 6(a)  and 6(b)). Nearly all of these downregulated DEGs shared the same results in Ala, Deh, and Mix groups.
At the same time, we also analyzed the upregulated expression of genes Blvrb, Prdx1, Cat, Srxn1, Slc48a1, and Sqstm1 by RT-qPCR. Stimulation of BV2 cells with LPS had no significant difference in the expression of Blvrb, Prdx1,

Discussion
In this study, we applied RNA-seq to systematically analyze the transcriptome of BV2 cells treated with Ala, Deh, and the Mix (mixture of Ala and Deh) after LPS stimulation. e RNA-seq gene expression profiles showed that the Deh group (957 DEGs) had significantly more DEGs than the Ala group (378 DEGs); however, the Mix group (1718 DEGs) had more DEGs than the Deh group (Figure 3). According to |log2FC| ≥1, FDR ≤0.05, and FPKM ≥100, top 9 downregulated and top 13 upregulated DEGs in both Ala and Deh were screened out. Among the downregulated DEGs, chemokines (Ccl7, Ccl2, and Cxcl10), interleukin (Il1β), tumor necrosis factor (Tnfα ), colony stimulating factor (Csf3), and Nos2 enzyme were all proinflammatory factors. And most of the upregulated DEGs had antioxidant effects, such as Sqstm1, Srxn1, Prdx1, and Cat.
C-C motif chemokine ligand 2 (Ccl2) was upregulated in MCAO/R model rat brain tissue; downregulation of Ccl2 inhibited the inflammatory response and decreased the ischemic infarct area and also inhibited Ccl7, Ccr2, Cxcl16, and Tnfα expression [25]. ymoquinone was one of the major compounds found in Nigella sativa and had antiinflammatory effects that can attenuate the Ccl2, Csf3, Cxcl10, Il6, and other cytokines/chemokines released by BV2 microglia cells after stimulation of LPS [26]. C-C motif chemokine ligand 7 (Ccl7), also called monocyte chemotactic protein-3 (MCP-3), had been demonstrated to influence neutrophil chemotaxis [27]. Upregulation of Ccl7 recruits monocytes to the injury site, mediating local inflammatory responses that may associate with disease pathogenesis and clinical symptoms [28]. Colony stimulating factor 3 (granulocyte) (Csf3 or G-CSF) was upregulated by LPS stimulation in BV2 cells. In response to bacterial infections and cell-mediated immune responses, the production of Csf3 increases dramatically. Csf3, as a proinflammatory cytokine, can pass through the blood-brain barrier (BBB) and plays a neuroprotective role in stroke [29]. Csf3 had no significant regulatory role in the unactivated resting microglia, but LPS-stimulated microglia pretreated with Csf3 could significantly inhibit the NO expression level [30]. Subcutaneous injection of recombinant human Csf3 in spinal cord-hemisectioned mice can recruit microglia to the injured site, and Csf3 can inhibit the expression of proinflammatory factors and promote the expression of neurotrophic factors [31].
RNA sequencing found that Ala, Deh, and Mix mainly downregulate the proinflammatory genes and upregulate antioxidant genes, respectively. e results showed that these compounds may contribute to the neuroprotective effects through anti-inflammatory and antioxidant activities. e effect of group Mix was more significant than that of the other two groups Ala and Deh, and its effect was not simply the sum of the effects of Ala and Deh (Table 2, Figures 6 and  7). e Mongolian medicine Eerdun Wurile is a mixture of Ala, Deh, and other small molecules, so its effect is also not a simple single effect addition.
GO terms were grouped in three ontologies: biological process, molecular function, and cellular component. In the top 20 GO terms, compared with Ala and Deh, DEGs in Mix were mainly enriched in molecular function ontology, but DEGs in each group were mainly enriched in protein binding. In the biological process, the DEGs were mainly enriched in the term of immune system process in the Ala group and Deh group, while a large number of DEGs were mainly enriched in cell cycle in the Mix group (Figures 4(d)-4(f)). is indicated that the Mix had different effects from Ala and Deh in the biological process. e KEGG pathway analysis indicated that the DEGs were most enriched in NOD-like receptor signaling pathway, TNF signaling pathway, and cytokine-cytokine receptor interaction. DEGs in all three pathways were mainly chemokines and cytokines, such as Ccl2, Ccl12, Cxcl2, Il6, and Il1β. Although each group had similar enrichment pathways, DEGs were very different, indicating that Ala, Deh, and Mix trigger different effects. Compared with the Ala group and Deh group, the DEGs in the Mix group were differently enriched in DNA replication (pathway ID:03030), cell cycle (pathway ID:04110), apoptosis (pathway ID:04210), and cell cycle-yeast (pathway ID:04111) ( Figure 5). DEGs in both DNA replication pathway and cell cycle pathway were Mxm2-7 and Pcna. Proliferating cell nuclear antigen (Pcna) was a cell cycle marker protein, which is well known to be an important part of eukaryotic chromosomal DNA replication and repair [40]. e minichromosome maintenance (Mcm) complex is in a ring shape, containing individual Mcm polypeptides (Mcm2-7). It was essential to initiating DNA replication and replication fork progression. During TBI, Mcm3 expression will be upregulated and promotes neuronal apoptosis [41].
e RNA-seq results showed that Mix significantly downregulated the expression of Mcm3 in LPS-stimulated BV2 cells.
To validate DEGs data, 8 inflammation-related cytokines and chemokines and 6 oxidation-related genes were selected for quantitative real-time PCR analysis. Nearly all of these down-and upregulated DEGs shared the same results in Ala, Deh, and Mix groups. RNA-seq and RT-qPCR results demonstrated that the Mix had the strongest downregulation effect on the proinflammatory cytokines including Csf3, Ccl7, Ccl2, Il1rn, Il1β, Cxcl10, Tnfα, and Nos2. At the same time, the Mix had remarkably increased the antioxidant genes including Blvrb, Prdx1, Cat, Srxn1, Slc48a1, and Sqstm1.
Traditional medicines such as Chinese and Mongolian medicines comprised of multiple components. e therapeutic mechanism of such medicine compound recipe relayed on the multiple biologically active molecules, which often simultaneously modulate multiple targets through multiple cellular pathways. In this experiment, RNA-seq was used to analyze the synergistic regulation effect of Ala and Deh (Mix) in BV2 microglial cells. Ala and Deh were found in Inula helenium L. and Myristica fragrans Houtt., the two main components of EW. We found that the synergistic effect of bioactive molecules is not simply the overlaying but also generate novel regulation effects that differ from both single molecules. Our data provide important evidence for further research on the therapeutic mechanism of traditional medicine including EW.

Data Availability
e data used to support the findings of this study are included within the Supplementary Materials file.