Adonis coerulea Maxim. was collected from Tianzhu County in Gansu Province, China (37°11.4′ N latitude, 102°46.1′ E longitude, 3,200 m), in August 2019, and the raw material was identified by Prof. Zhigang Ma, Pharmacy College of Lanzhou University, China. A voucher specimen with accession number ZSY112-T1 was submitted to the Herbarium of Lanzhou Institute of Husbandry and Pharmaceutic Sciences, CAAS (Lanzhou, China).

The primary mouse macrophage RAW 264.7 cells were cultured in medium prepared with 10% fetal bovine serum (FBS) and 90% Dulbecco’s modified eagle medium (DMEM) under a humidified incubator of 5% CO₂ at 37°C. The cytotoxicity of the methanol extract of ACAP against RAW 264.7 cells was evaluated using the ZETA cell counting kit (ZETA Life, United States). After incubating RAW 264.7 cells (5 × 10⁴ cells/well) in 96-well plates for 24 h, ACAP (10–1,000 μg/ml, 10 μl) was added and incubated again for 24 h. Then, the cell counting kit-8 (CCK-8, 10 μl) was added and co-cultured for 30 min, and the absorbance was measured at 450 nm. Dimethyl sulfoxide (DMSO) (0.1%) was used as a control.

The anti-inflammatory activity was evaluated by determining the inhibitory effect of ACAP on the production of inflammatory cytokines in RAW 264.7 cells, which was induced by lipopolysaccharide (LPS). 500 μl of RAW 264.7 cells (5 × 10⁴ cells/well) were incubated in 48-well plates for 24 h, and then 500 μl of ACAP (10–100 μg/ml) and dexamethasone (DEX, positive agent) (10 μg/ml) were added and cultured for 1 h again, respectively. After adding LPS (2 μg/ml) for 24 h, the cells were collected to determine the levels of nitric oxide (NO), interleukin-1β (IL-1β), and tumor necrosis factor-a (TNF-a) using enzyme-linked immunosorbent assay (ELISA) kits from Nanjingjiancheng Bio (NJJCBIO, China) (15).

First, 20 compounds identified from ACAP were selected for predicting the possible drug targets using Swisstargetprediction (16), which were filtered by probability ≥ 0.12 and checked using the Uniprot database defined as Homo sapiens. Meanwhile, the “anti-inflammatory” was used as terms to search the related genes from the Genecards database (17), and the intersection between the anti-inflammatory-related targets and the predicted targets of ACAP was picked as common targets.

Subsequently, these common targets were submitted to the STRING 11.0 database (18) to construct a protein-protein interaction (PPI) network with a minimum required interaction score more than 0.4 confidence; the hub targets and core compounds were performed by constructing the “Compounds-Targets” network. Finally, the hub targets were imported into the Enrichr database (19) to perform functional annotation and enrichment analysis. Gene Ontology (GO) terms and KEGG pathways with p < 0.05 were conducted.

Molecular docking between identified targets and active compounds of ACAP was performed using the DOCK 6.9 software in the Yinfo Cloud Platform. The grid score (kcal/mol) was calculated based on the best pose.

The anti-inflammatory activities of compounds were evaluated by determining the inhibitory effect of ACAP on NO in RAW 264.7 cells induced by LPS. Cells (5 × 10⁴ cells/well, 500 μl) were incubated in 48-well plates for 24 h, and then 500 μl of linolenic acid, licochalcone A, vitexin, isoorientin, orientin, caprylic acid, L-phenylalanine (50 μg/ml), and DEX (5 μg/ml) were added and cultured for 1 h again, respectively. After adding LPS (5 μg/ml) for 24 h, the cells were collected to measure the level of NO using ELISA kits (NJJCBIO, China) (15).

Cells (5 × 10⁴ cells/well, 500 μl) were incubated in 48-well plates for 24 h, and then 500 μl of ACAP (10–100 μg/ml) and DEX (positive agent) (10 μg/ml) were added and cultured for 1 h again, respectively. After adding LPS (2 μg/ml) for 24 h, the cells were collected to determine the levels of peroxisome proliferators-activated receptor (PPAR-γ) using mouse peroxisome proliferators-activated receptor ELISA) kits from Nanjingjiancheng Bio (NJJCBIO, China).

After treating with ACAP for 1 h, LPS (2 μg/ml) was added to stimulate RAW 264.7 cells for 24 h. The protein of cells was extracted and then separated on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% bovine serum albumin for 1 h at room temperature and incubated overnight. Antinuclear factor-κB (IκB) alpha, anti-IκB alpha (phospho S36), anti-NF-κB p65, and anti-NF-κB p65 (phospho S536) were purchased from Abcam (Shanghai, China) for the immunoblotting, and the relative protein expression was quantified compared with the β-actin level.

After 24 h of treatment with ACAP, H₂O₂ (0.6 mM) was used to stimulate RAW 264.7 cells for 24 h; the cell culture was replaced with JC-1 staining solution. After incubating at 37°C for 30 min in darkness and washed with PBS, the fluorescence was photographed using an LSM-800 with Airyscan using the JC-1 assay kit (Solarbio, China). For red fluorescent of J-aggregates and the fluoresce green of J-monomers, the wavelength of excitation/emission was set as 525/590 nm and 490/530 nm, respectively (20).

After 24 h of treatment with ACAP, H₂O₂ (0.6 mM) was used to stimulate RAW 264.7 cells for 24 h, and then cells were incubated with 2 mM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (NJJCBIO, China) at 37°C for 30 min in darkness and photographed (excitation/emission 500/525 nm) using an LSM-800 with Airyscan. The fluorescence index was calculated for measuring the ROS production.

Results are expressed as mean ± standard deviation. Data were analyzed by one-way ANOVA, followed by the Dunnett’s test when the data involved three or more groups using GraphPad Prism 8.0.2. Differences were considered statistically significant at p < 0.05.

Subsequently, the chemical compositions of ACAP were analyzed using UPLC- MS/MS, and the LC/MS chromatographic peak of both negative and positive ions were presented in Figure 1. The data on the retention time, molecular ion, main MS² fragments, tentative chemical compounds, and molecular formula of ACAP were described in Table 2.

In Figure 1, we can see that about 30 compounds were observed in the total ion chromatogram. By comparing the mass spectrometric data of the literature and the natural product library, only eighteen compounds were identified, namely, flavonoids, amino acids, fatty acids, cardiac aglycone, alkaloids, terpenoids, phenols, and others. Other unknown compounds should be identified further to found the possible active ingredients. Among the identified compounds, most of the compounds belong to flavonoids, including licochalcone A, isoorientin, orientin, luteolin-5-glucuronide, 5′-hydroxypuerarin, vitexin, norwogonin-7-O-glucuronide, 3-rha-7-rha-quercetin, and acacetin-7-O-rutinoside (32); one amino acid, phenylalanine (33), three fatty acids, caprylic acid, linolenic acid, and linoelaidic acid (34, 35) were also detected from ACAP. In addition, one alkaloid magnoflorine (36), one terpenoid bryoamaride (37), one cardiac aglycone strophanthidin (38), and one coumarins 4-methylumbelliferyl glucuronide (39) were identified from this species. Isoorientin, orientin, and strophanthidin were also found by previous reports (38, 40). In addition, this result was consistent with the result of the total phenol and flavonoid analyses, and the content of flavonoids in ACAP was more than the content of phenols. Flavonoids, widely existed in plants, possessed the wide range of biological activities, such as antioxidant, anti-inflammatory, antibacterial, and immune regulation (41). Hence, we hypothesized that ACAP may have the possible anti-inflammatory and antioxidant activities.

The safety of ACAP was investigated by evaluating the cytotoxicity of ACAP against RAW 264.7 cells. Result showed that at the concentrations of 0–1,000 μg/ml, ACAP presented the safety in vitro. Even at 1,000 μg/l, the cell viability was more than 50% (Figure 2A).

Macrophages stimulated by LPS and other foreign substances would produce and release the proinflammatory cytokines, which engaged into the process of inflammatory damage (42). In this study, the anti-inflammatory activity of ACAP in vitro was investigated by determining the inhibitory effect on the production of inflammatory cytokines.

As a well-known inflammatory cytokines, NO is released by activated macrophages through the formation of peroxynitrite with superoxide anion and inflammatory response. Figure 2B showed that after the stimulation by LPS, the content of NO in RAW264.7 cells (81.40 μM) was rapidly increased compared with control group (12.41 μM, p < 0.001); however, the release of NO was significantly inhibited after the treatment of ACAP; the contents were 72.09 μM for 10 μg/ml, 56.59 μM for 25 μg/ml, 50.39 μM for 50 μg/ml, and 41.09 μM for 100 μg/ml (p < 0.001). In Figure 2C, we could see that after the treatment of ACAP, the levels of TNF-a in RAW 264.7 cells also were decreased compared with the model group in a dose-dependent manner, which plays an important role in inflammation formation. In addition, IL-1β mainly participated in the immune response and tissue repair in response to inflammatory diseases; the result showed that ACAP could decreased the inflammatory response induced by LPS by reducing the produce of IL-1β in a dose-dependent manner (Figure 2D). These results showed that ACAP presented the significant anti-inflammatory activity in a dose-dependent manner by decreasing the production of proinflammatory cytokines.

A network pharmacology was performed to explore the potential mechanism of action of ACAP. Eighteen compounds identified from ACAP was used as a candidate to collect the potential targets. In total, 48 targets acted with 12 compounds were screened out from the SWISSTARGET database, and 3,512 targets related anti-inflammatory were obtained; however, only 39 common targets were filtered out between inflammatory targets and compounds targets for the further study (Figure 2E).

Subsequently, a PPI network of the common targets was constructed using the String database, the total of 45 nodes and 152 edges were obtained, respectively, and the average degree of node was 6.76. The targets PPARA, EGFR, TNF, and PPARG with more edges and nodes were located in the core hub (Figure 2F). A compound-target network was established and constructed to obtain the potential mechanism, and 39 putative hub targets and 12 core compounds were identified. Among of compounds, linolenic acid and linoelaidic acid have the highest degree value (16), and the target AKR1B1 has the high degree of interaction with other targets and compounds (Figure 2G).

GO analysis showed that these genes were enriched in the regulation of inflammatory response, negative regulation of lipid localization, and negative regulation of lipid storage and other biological processes and participated in cellular components, such as membrane microdomains, membrane raft; these genes were involved in cytokine receptor binding, cytokine activity, carboxylic acid binding, protease binding, and others were involved in molecular functions. This result suggested that the targets of SRP against inflammatory related to regulation of the inflammatory response and cytokines. KEGG pathway analysis showed that the potential targets of active compounds in ACAP were mainly enriched in PPAR (peroxisome proliferators-activated receptors) signaling pathway, T-cell receptor signaling pathway, neuroactive ligand-receptor interaction, AMPK signaling pathway, and others (Figure 2H). These results indicated that SRP presented antiliver inflammatory activity by regulating the PPAR-mediated signaling pathways, and further studies were performed to prove this hypothesis.

To verify the result of network pharmacology and find the active compounds of SRP, the anti-inflammatory activities were evaluated using the NO assay. As shown in Figure 3A, all compounds from ACAP significantly inhibited the NO production of RAW 264.7 cells induced by LPS (p < 0.001). Linolenic acid presented the best anti-inflammatory activity with the NO content of 65.34 μM, followed by licochalcone A (73.76 μM), vitexin (91.26 μM), and caprylic acid (95.01 μM). Orientin, isoorietin, and acacetin-7-O-rutinoside have the weak inhibitory effect on NO, whereas, NO contents of the control and DEX were 57.51 μM and 93.76 μM, respectively (Figures 3A,B). Considering that the oral bio-availability of only linolenic acid and licochalcone A were more than 40%, these results indicated that above compounds have the synergistic effect against anti-inflammatory activity for SRP, linolenic acid, and licochalcone A as active compounds.

To prove the result of network pharmacology, the molecular docking between the targets AKR1B1 (PDB 5OUK) and PPAR (PDB 3VI8) and active compound linolenic acid and acacetin-7-O-rutinoside were conducted to prove their affinity and find the binding core, respectively. The result showed that linolenic acid bonded to with Leu (212, 300), Ser (214), Lys (262), Ile (260), and Trp (111, 219) of AKR1B1 with the high affinity, the grid score, and the internal energy of -75.79 kcal/mol (Figure 3C). Acacetin-7-O-rutinoside has the high affinity to the target PPAR with the grid score of -87.66 kcal/mol, it could bind to Tyr (334), Ala (256), Thr (279), and Cys (275) with hydrogen bond, to Val (332), Ile (241), and Ala (250) with hydrophobic interaction (Figure 3D). This result indicated that two active compounds played the potential anti-inflammatory activity via PPAR and AKR1B1, respectively.

PPARs are ligand-activated transcription factors of nuclear hormone receptor superfamily, which play essential roles in the regulation of cellular differentiation, development, and metabolism and tumorigenesis. As one of the subtypes, PPAR-γ participated in the inflammatory response via the regulation of NF-κB. The activated PPAR-γ is translocated into the nucleus to bind with NF-κB, and then the NF-κB-PPAR-γ complex was formed, which cannot bind to the promotor region of DNA; thereby, gene expressions of proinflammatory mediators were suppressed (43). As shown in Figure 3E, ACAP could increase the level of PPAR-γ in RAW 264.7 cells induced by LPS in a concentration-dependent manner, especially for high concentration (100 μg/ml). This result proved the result of the network pharmacology assay. Considering that many fatty acids could bind to PPAR-γ, this result indicated that ACAP played the anti-inflammatory activity by activating PPAR-γ and then inhibiting the gene expressions of proinflammatory mediators, such as linolenic acid.

The IkappaB kinase (IKK) complex was activated and led to the phosphorylation and degradation of the inhibitory IκB proteins and then stimulate activation of NF-κB to regulate the production of proinflammatory factors, including NO, TNF-α, IL-1β, and IL-6 (43). Hence, NF-κB axis is important to the expression of proinflammatory genes, and the modulation was thought as a good strategy to the treatment of inflammatory diseases by inhibiting proinflammatory gene expression. To prove the result of network pharmacology and explore the potential mode of action, the expressions of phosphorylation of IκB (p-IκB)/IκB and the phosphorylation of NF-κB (p-NF-κB)/NF-κB were investigated. As shown in Figures 4B,C, compared with the mode group, the rations of p-IκB/IκB and p-NF-κB/NF-κB of RAW 264.7 cells induced by LPS were decreased after the treatment of ACAP in a dose-dependent manner, especially for p-IκB/IκB (p < 0.001) (Figures 4A–C).

Macrophages stimulated by H₂O₂ would cause the oxidant stress by elevating ROS, MDA, and attenuating antioxidant enzymes. SOD and CAT provide first-line cellular protection contributing to prevent cellular damage and maintain a balance between free radical production and oxidative stress after stimulated by oxidative stress (44). After the treatment of ACAP, the activity of SOD in RAW 264.7 cells was significantly activated compared with the model group in a dose-dependent manner (p < 0.01). At the concentrations of 100 μg/ml, the SOD activities were 47.12 U/mg Prot (p < 0.001), respectively, similar to the positive agent Vc of 50.68 U/mg Prot at 10 μg/ml (p < 0.001) (Figure 4D). At the high concentrations of 50 and 100 μg/ml, the CAT activity also was significant increased compared with the model group (p < 0.001) (Figure 4E). As well as the similar dose-dependent manner on CAT, ACAP decreased the elevated production of MDA in cells stimulated by H₂O₂ at the high concentrations (p < 0.001) (Figure 4F), which would be released when responding to oxidant stress. These results indicated that ACAP has the cellular antioxidant activity by activating the SOD and CAT activities and scavenging free radicals from macrophages induced by H₂O₂.

In this test, the red fluorescence was seen as an aggregated pattern of energized mitochondria, and the green fluorescence was presented as a monomeric pattern for depolarized mitochondria. In Figure 5A, after the treatment of ACAP, the intensity of red and green fluorescence was reversed in a dose-dependent manner and showed higher red fluorescence and lower green fluorescence; however, cells induced by LPS presented the remarkably lower red fluorescence and higher green fluorescence. This result indicated that ACAP could improve the MMP depletion induced by LPS.

The oxidant response to exogenous substance would increase cellular ROS production (45). Hence, the cellular ROS production in RAW 264.7 cells was evaluated by determining the intensity of green fluorescence, which was stained by DCFH-DA. Compared with the control group (FI 464.05), the fluorescence intensity (FI) in the model group was significantly enhanced with the value of 1,583.13. However, after the treatment of ACAP, the green fluorescence become vague, and the intensities were decreased in a dose-dependent manner. The inhibition rates were 43.25% for 100 μg/ml (p < 0.01), 25.63% for 50 μg/ml, and 14.89% for 25 μg/ml. This result illustrated that ACAP inhibited the ROS production and accumulation of RAW264.7 cells induced by H₂O₂ (Figure 5B). Combined with the above result, we thought that ACAP played the anti-inflammatory and antioxidant activities by inhibiting PPAR-γ mediated NF-κB signaling pathway (Figure 6). The further studies should be carried out to prove this mode of action of ACAP and its active compounds.