Orostachys japonicus extract inhibits the lipopolysaccharide-induced pro-inflammatory factors by suppression of transcription factors.

Abstract Orostachys japonicus (O. japonicus) was extracted with ethanol (EtOH) and sequentially separated with organic solvents, including n‐hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc), n‐butanol (BuOH), and water (H2O). All the fractions were confirmed for anti‐inflammatory activity in an inflammatory condition. The DCM fraction showed the highest anti‐inflammatory ability. Here, we examined the effect of DCM fraction and investigated the intracellular signaling pathways in LPS‐stimulated RAW 264.7 macrophage cells. The DCM fraction significantly inhibited the mRNA levels of pro‐inflammatory mediators and cytokines including iNOS, COX‐2, IL‐1β, IL‐2, IL‐6, and IP‐10 in LPS‐stimulated cells. Also, the treatment of DCM fraction excellently reduced the expression of the proteins of AP‐1 (phospho‐c‐Jun and phospho‐c‐Fos) and phospho‐IRF3 as transcription factors. As a result, it suppressed LPS‐induced inflammatory mediator and cytokines via inhibition of transcription factors. In conclusion, our data demonstrated that DCM fraction has a strong anti‐inflammatory activity that improves the inflammatory state.


LEE
Orostachys japonicus is a polygamous plant in the family Crassulaceae that has been used as a medicinal herb for fever, hemostasis, antidote, inflammation, and cancer (Kim, Choi, Park, Lee, & Jung, 2009;Kwon & Han, 2004;Ryu, Lee, Lee, & Lee, 2012;Yoon et al., 2009). The previous studies have reported that O. japonicus has beneficial ingredients, such as flavonoids, triterpene, and galactic acid (Park, Han, Park, Choi, & Choi, 2005). Thus, dried O. japonicus was extracted with six kinds of organic solvents. The fractionated O. japonicus was expected to active various physiological processes, including inflammation.
In this study, we confirmed the effective O. japonicus fraction for treating the LPS-induced inflammatory condition. Furthermore, we wanted to find out the effect of O. japonicus on intracellular signals of cytokines and transcription factors in LPS-stimulated macrophage cells.

| Cell culture and experimental reagents
Mouse macrophages (RAW 264.7 cells) were obtained from the Korean Cell Line Bank (KCLB). Cell was cultured on complete DMEM media added with 1% antibiotics (50× penicillin and streptomycin) and 10% fetal bovine serum (FBS) (Welgene) at 37°C in a 5% CO 2 . The macrophages were maintained to culture every 2-3 days at 1:6 split ratios. Rabbit primary antibodies against phospho-c-Jun, phospho-c-Fos, phospho-IRF3, and GAPDH (housekeeping gene) were ordered from Cell Signaling Technology Inc.. HRP-conjugated second antibody was purchased from BD Pharmingen™ (BD Biosciences).

| Fractionated Orostachys japonicus with organic solvents
Fractionated O. japonicus was supplied from a farm in Miryang (Geobugiwasong Ltd.). The O. japonicus was separated using organic solvents, and the extract method was described in the previous studies Lee, Kim, & Lee, 2018;Ryu et al., 2012). Each fraction removed the solvents by evaporator at 40°C to dryness. It was lysed in dimethyl sulfoxide (DMSO) and retained in a frozen state (Lee, Bilehal, et al., 2013).

| Cell proliferation analysis
Cell viabilities were confirmed with an MTS assay kit (Promega Corporation) in the protocol. RAW 264.7 cells were incubated with serial doses (0, 25, 50, 75, and 100 μg/ml) of organic solvents for 24 hr. After the reaction, 20 μl of solution of cell proliferation assay was added and formed a formazan for 4 hr. The results were measured of absorbance at 490 nm using a FilterMax F5 microplate reader (Molecular Devices).

| Reverse transcription polymerase chain reaction (RT-PCR)
Cells were pretreated with solvent fractions of O. japonicus for 2 hr and then LPS-induced inflammation for 12 hr. Total RNAs were separated with the cells of 6-well plate using the Trizol™ reagent (Invitrogen). The concentrations of the total RNA were measured at 260 nm by a FilterMax F5 microplate reader (Molecular Devices); 2 μg RNA and 1 μg/μl oligo(DT) were added to AccuPower Reverse Transcription PreMix tube for the cDNA synthesis (Bioneer). The amplification of the target gene was performed using manufactured primers of forward and reverse in the PCR cycler. The primer sequences and conditions used in the PCR cycler are arranged in Table 1. After PCR, the products were transferred on 1.5% agarose gels and exposed the ethidium bromide (EtBr) in the electrophoresis system. The band density was determined and visualized using the Davinch-Chemi™ imaging system (Davinch-K).

| Statistical analysis
All experiments were carried out more than three times. The data were reported as the mean ± standard deviation (SD) and analyzed by SPSS (version 21). A difference between the experimental groups was significantly expressed at p < .05 and p < .01.  (Lee, Bilehal, et al., 2013;, and it will be applicable to the prevention and treatment of various diseases.

| RE SULTS AND D ISCUSS I ON
Among them, DCM fraction from O. japonicus (OJD) showed the best anti-inflammatory effect on LPS-stimulated cells. We used a gas chromatography-mass spectrometry (GC-MS) system to examine the active components in the DCM fraction. As a result, 11 peaks of them were hard to identify, but 3 peaks were identified as kaempferol (7.76%), quercetin (6.51%), and campesterol (53.53%) .
Furthermore, research on the ingredients of O. japonicus will continue.

| Effect of the Orostachys japonicus solvent fractions on cell viability
To assess the cytotoxic effect of the solvent fractions, the cells

F I G U R E 2 Inhibitory effect of the Orostachys japonicus solvent fractions on pro-inflammatory mediators and cytokines.
Macrophage cells were pretreated with solvent fractions (100 μg/ ml) for 2 hr and LPS-stimulated inflammation for 12 hr. Expression of iNOS, COX-2, IL-1β, IL-2, IL-6, and IP-10 was analyzed by RT-PCR affected by any of the conditions. Based on this, the highest concentration was selected to be 100 μg/ml (Figure 1).

| Effect of the Orostachys japonicus solvent fractions on pro-inflammatory mediators and cytokines
The inflammatory response activates macrophages and pro-inflammatory mediators, and cytokines are expressed, such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1β, IL-2, IL-6, and interferon-gamma inducible protein 10 kDa (IP-10). Their overproduction of these molecules is a common marker of the inflammatory response, resulting in various adverse effects (Park et al., 2017;Yao et al., 2016). Inhibiting the expression in these inflammatory mediators is important to verify the anti-inflammatory activity of a natural substance. To investigate whether O. japonicus induces anti-inflammatory mediators and cytokines, the expression of iNOS, COX-2, IL-1β, IL-2, IL-6, and IP-10 was examined by RT-PCR.
The cells were pretreated to the organic solvent fractions (100 μg/ ml) for 2 hr and LPS-induced inflammation for 12 hr. As shown in

| Effect of the Orostachys japonicus solvent fractions on transcription factors
Lipopolysaccharide binds TLR4, leading to the activation of intracellular signaling pathways of two types, such as the MyD-88-and TRIFdependent pathways. Both of these pathways play important roles in regulating nuclear translocation of inflammatory factors, such as NF-κB, AP-1 (p-c-Jun and p-c-Fos), or IRF-3. Pro-inflammatory mediators and cytokines were expressed in LPS-stimulated cells after the transcription factors are translocated to the nucleus. Inhibiting translocation into the nucleus is an important mechanism to regulate inflammation (Kim, Han, Kil, Seo, & Jin, 2019;Park et al., 2017;Takeda & Akira, 2005). To conform for inhibits of solvent fractions, cells were pretreated with the highest concentration of solvent fractions (100 μg/ml) for 2 hr and LPS-induced inflammatory for 1.5 hr. As shown in Figure 4, the protein levels of AP-1 (p-c-Jun and p-c-Fos) and p-IRF-3 were the lowest in the DCM fraction.
We also detected a significant decrease in a dose-dependent manner ( Figure 5). These observations suggest that the DCM fraction is a negative regulator of LPS-induced nuclear translocation of AP-1 and IRF-3 in macrophage cells. AP-1 and IRF-3 are regarded as pivotal factors in the regulation of inflammation by producing pro-inflammatory mediators and cytokines, such as iNOS, COX-2, IL-1β, IL-2, IL-6, and IP-10.

| CON CLUS ION
In

CO N FLI C T O F I NTE R E S T
The author declares no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or vertebrate animal.