Lonicera japonica Thunb. Ethanol Extract Exerts a Protective Effect on Normal Human Gastric Epithelial Cells by Modulating the Activity of Tumor-Necrosis-Factor-α-Induced Inflammatory Cyclooxygenase 2/Prostaglandin E2 and Matrix Metalloproteinase 9

Gastric inflammation-related disorders are commonly observed digestive system illnesses characterized by the activation of proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α). This results in the induction of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PEG2) and matrix metallopeptidase-9 (MMP-9). These factors contribute to the pathogenesis of gastric inflammation disorders. We examined the preventive effects of Lonicera japonica Thunb. ethanol extract (Lj-EtOH) on gastric inflammation induced by TNF-α in normal human gastric mucosa epithelial cells (GES-1). The GES-1 cell line was used to establish a model that simulated the overexpression of COX-2/PGE2 and MMP-9 proteins induced by TNF-α to examine the anti-inflammatory properties of Lj extracts. The results indicated that Lj-EtOH exhibits significant inhibitory effects on COX-2/PEG2 and MMP-9 activity, attenuates cell migration, and provides protection against TNF-α-induced gastric inflammation. The protective effects of Lj-EtOH are associated with the modulation of COX-2/PEG2 and MMP-9 through the activation of TNFR–ERK 1/2 signaling pathways as well as the involvement of c-Fos and nuclear factor kappa B (NF-κB) signaling pathways. Based on our findings, Lj-EtOH exhibits a preventive effect on human gastric epithelial cells. Consequently, it may represent a novel treatment for the management of gastric inflammation.


Introduction
Lonicera japonica Thunb.(Lj) is a Chinese herbal plant (homology of medicine and food) of which the flower, seeds, and leaves have been used for medicine since ancient times.Many studies have been conducted to evaluate the activity of Lj extract, and its various effects on human health have been established, including antioxidative, antibacterial, antiinflammatory, antiviral, anticancer, and various other activities [1].The dried flower buds of Lj may be prepared as tea which is known for its detoxifying and soothing effects [2].
Recent studies have provided evidence suggesting a progressive rise in gastric inflammation-related disorders, which may be attributed to a multitude of factors, such as an aging population, the prevalence of chronic pain, and the increased use of nonsteroidal anti-inflammatory drugs (NSAIDs) or diverse chemotherapeutic medications.These factors contribute to a rapid escalation of gastric inflammation-related disorders, which can worsen gradually over time [3,4].
Gastric inflammation-related disorders arise from the chronic erosion of gastric mucosal epithelial cells, which surpasses the regenerative capacity of these cells.This imbalance leads to the manifestation of gastric inflammation, fibrosis at the site of injury, and thinning of the gastric wall and may result in complications, such as gastric bleeding, gastric perforation, or the development of gastric cancer (GC) [5].
The etiology of gastric inflammation includes impaired or recurrent inflammation of the gastric mucosal epithelial tissue, which culminates in the activation of cytokines and chemokines.This subsequently results in the activation and infiltration of macrophages, neutrophils, monocytes, and other immune cells into the gastric tissue.The immune cells produce proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukins such as IL-1, IL-6, IL-8, and IL-10, and cell adhesion proteins like circular dichroism 44 (CD44) and intercellular adhesion molecule-1 (ICAM-1).The aforementioned factors serve as triggers for the production of inflammatory proteins, including cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9), within the mucosal lining in GC.The pathophysiological features of gastric inflammation arise from stimulation by immune cells, such as neutrophils, monocytes, and macrophages, and their infiltration into the gastric tissue.This leads to the development of inflammatory responses in the gastric mucosal epithelial cells, subsequent damage to the gastric mucosa, and the degradation of gastric secretory glands, among others [6][7][8].
MMP-9 contributes to the development of various human malignancies by facilitating wound healing, cell migration, angiogenesis, and tumor progression through collagen IV degradation in the basement membrane and extracellular matrix [20].The upregulation of MMP-9, mediated by the nuclear factor kappa B (NF-κB) signaling, may promote gastric inflammation and actively contribute to the progression of GC [21].Moreover, the findings in our previous study indicated that under normal conditions, quercetin exerts protective effects in GES-1 cells by inhibiting TNF-α-induced MMP-9 upregulation [22].
In gastric mucosal epithelium cells, Lj has been shown to exhibit gastroprotective properties following TNF-α-induced damage.However, the detailed underlying mechanisms are still unknown.Here, we elucidated the potential preventive properties of Lj on gastric mucosal epithelial cells in response to TNF-α-induced damage, with a specific focus on determining its effects on COX-2/PGE 2 and MMP-9 signaling pathways.We used GES-1 cells under normal conditions to assess the molecular processes and immune-inflammatory reactions regulated by TNF-α.

Preparation of Lonicera japonica Thunb. Extracts
The dry herb of Lj was provided by Sheng Chang Pharmaceutical Co., Ltd.(Taipei, Taiwan), where the Lj powder was extracted using a Dionex™ ASE™ 350 extractor (Thermo Fisher Scientific Inc., Foster City, CA, USA).Lj powder (5 g) was placed into 66 mL container equipped with a stainless-steel frit and a cellulose filter at the bottom.Extraction was carried out using H 2 O or 95% EtOH as the solvent at a temperature of 100  C until use.

Cell Counting Kit-8 (CCK-8) Assay
GES-1 cells were seeded into 96-well plates with a cell density of 5000 cells per well.The cells were incubated for 24 h in a humidified incubator containing 5% CO 2 .Lj extracts were diluted to prepare a range of concentrations (0, 0.1, 0.5, 1, 5, or 10 mg/mL), which were added to the cells and incubated for another 24 h.After adding 10 µL of CCK-8 kit reagent (MedChemExpress Ltd., Monmouth Junction, NJ, USA) to each well and incubating at 37 • C for 1 h, the cell density was measured using a microplate reader at 450 nm (SpectraMax i3; Kelowna International Scientific, NewTaipei, Taiwan) [24].To evaluate cell viability, the ratio of viable cells to the control group was quantified.

Quantitative Reverse Transcription-PCR (qRT-PCR)
GES-1 cells were seeded into 9 cm dishes and incubated at 37 • C for 24 h.The cells were treated with TNF-α (30 ng/mL) for 0, 16, 20, or 24 h.Total RNA was isolated from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA concentration was determined using a Nano100 Micro-Spectrophotometer (CLUBIO; Taipei, Taiwan).cDNA synthesis was carried out using the iScript cDNA Synthesis Kit manufactured by Bio-Rad Laboratories, Inc (Hercules, CA, USA), and amplification was carried out using SsoFast EvaGreen Supermix and a thermal cycler (iCycler; Bio-Rad Laboratories, Foster, CA, USA).To measure the expression of MMP9 and COX2 mRNA in GES-1 cells, qRT-PCR was conducted by using GAPDH as the internal control [25] through the utilization of the CFX Connect Real-Time PCR system.The primer sequences were as follows: For COX2, the sense primer was 5 ′ -CGGTGAAACTCTGGCTAGACAG-3 ′ and the antisense primer was 5 ′ -GCAAACCGTAGATGCTCAGGGA-3 ′ .For MMP9, the sense primer was 5 ′ -AGTTTGGTGTCGCGGAGCAC-3 ′ and the antisense primer was 5 ′ -TACATGAGCGCTTCCGGCAC-3 ′ .For GAPDH, the sense primer was 5 ′ -ACAGTCAGCCG CATCTTCTT-3 ′ and the antisense primer was 5 ′ -GACAAGCTTCCCGTTCTCAG-3 ′ .The quantification of gene expression levels was carried out by ∆∆Ct methodology, whereby Ct denotes the average threshold cycle value.

Cell Migration Assay
GES-1 cells were seeded in 6-well plates and cultured until confluence.The medium was replaced with a serum-free RPMI medium and incubated for 24 h.The cell monolayer was incised with disposable cell scrapers and any dislodged cells were removed with PBS.Following pretreatment with Lj extracts (1 µg/mL) or inhibitors targeting TNFα (1 µM), NF-κB (BAY 11-7082) (10 µM), c-Fos (TSIIA) (10 µM), ERK1/2 (U0126) (1 µM), MMP-9 (MMP9i) (5 µM), or COX-2 (NS-398) (5 µM) for 1 h, serum-free medium with or without TNF-α (30 ng/mL) was added to each well.Image acquisition was carried out by microscopy using a digital camera (Olympus, Tokyo, Japan) at two time points: 0 and 24 h.For each time point, a set of four-phase images was acquired and the average of these images was normalized to the initial image recorded at 0 h.The normalized values were subsequently averaged across all experimental conditions.The data represent three distinct and separate experimental trials [24].

Statistical Analysis
The data represent a minimum of three separate trials and are presented as the mean ± standard error of the mean (SEM).Statistical analysis was carried out using a one-way ANOVA analysis by GraphPad Prism 6.0 computer software (GraphPad computer software, Inc., San Diego, CA, USA), supplemented by Tukey's test to determine significant differences among multiple groups.p < 0.05 was considered statistically significant.The statistical methodologies were evaluated by Dr. Jian-Hao Chen, who is affiliated with the Estat Statistics Consulting Company (Taipei, Taiwan).

COX-2 and MMP-9 Expression upon TNF-α Treatment in GES-1 Cells
The increased expression of MMP-9 through the NF-κB-dependent pathway has been implicated in the acceleration of gastric inflammation and the pathogenesis of GC, a condition characterized by poor survival [27][28][29].Potter et al. reported that a marked activation of COX-2 occurs in inflamed neoplastic sites or GC [17].By using qRT-PCR, COX-2, and MMP-9 mRNA were measured in normal GES-1 cells treated with TNF-α.After stimulation with TNF-α, a significant increase in COX-2 and MMP-9 mRNA levels was observed in a time-dependent manner at 16 and 24 h compared with the 0 h time point (p < 0.01 for all) (Figure 1A,B).To determine the relationship between TNF-α-activated COX-2 and MMP-9 in GES-1 cells, the cells were exposed to 30 ng/mL TNF-α for 0, 2, 4, 6, 16, or 24 h.Following treatment, COX-2 and MMP-9 protein levels were measured.As compared with the 0 h time point, TNF-α treatment (30 ng/mL) for 16 or 24 h resulted in the highest increase in COX-2 and MMP-9 expression (p < 0.05, p < 0.01 at 16 h, and p < 0.01, p < 0.01 at 24 h, respectively).The induction of COX-2 and MMP-9 by TNF-α occurred in a time-dependent manner (Figure 1C,D).The results indicate that COX-2 and MMP-9 expression in GES-1 cells are enhanced at both mRNA (transcriptional) and protein (translational) levels following TNF-α exposure.Next, we determined whether a TNF-α antagonist could suppress the expression of COX2 and MMP9 induced by TNF-α.The cells were further pretreated with a TNF-α antagonist at 0.5, 1, or 5 µM for 1 h followed by TNFα exposure.There was a marked reduction in the expression of TNF-α-induced COX-2 in GES-1 cells in a time-dependent manner after 16 and 24 h of incubation compared with the control (p < 0.05 for all at 16 h and p < 0.01 for all at 24 h, respectively) (Figure 1E).Similarly, over 16 h and 24 h, MMP-9 expression was also decreased considerably in GES-1 cells upon TNF-α antagonist treatment compared with the 0 h measurement (p < 0.05 for 1 µM TNF-α antagonist, p < 0.05 for 5 µM at 16 h and p < 0.01, for all at 24 h, respectively) (Figure 1E,F).One hour of exposure before addition of the TNF-α antagonist at different concentrations is effective in the shortest time for gastric anti-inflammatory response (Figure 1E,F).In this study, pretreatment with drugs for one hour could provide a preventive effect.
The transcription factors AP-1 and NF-κB play an important role in modulating the promoter region of the human COX-2 and MMP-9 genes, in which they engage in interactions with cytokines and growth factors [34,35].To determine the potential involvement of the above signaling pathways in decreased AP-1 promoter reporter activity caused by Lj-EtOH, a human AP-1 response element reporter assay was conducted.Treatment with Lj-EtOH led to a reduction in AP-1 reporter activity (Figure 3E).Taken together, the results indicate that Lj-EtOH decreases the expressions of TNF-α-induced MMP-9 and COX-2 in normal GES-1 cells through the TNFR-ERK1/2-c-Fos pathway.

The Effect of NF-κB on TNF-α-Stimulated COX-2 and MMP-9 Expression in Normal GES-1 Cells
Western blot analysis was carried out to determine whether Lj-EtOH, TNF-α antagonist, U0126, TSIIA, and BAY 11-7082 affected TNF-α-induced NF-κB (p65) phosphorylation.Thus, the phosphorylation of NF-κB (p65) was measured at various time intervals.Pretreatment with Lj-EtOH, TNF-α antagonist, U0126, TSIIA, and BAY 11-7082 resulted in the suppression of NF-κB phosphorylation (p-p65) upon TNF-α stimulation in a timedependent manner (Figure 4A).An immunofluorescence stain was used to determine the effect of Lj-EtOH on the translocation of NF-κB from the cytoplasm to the nucleus.After exposure to 30 ng/mL TNF-α for 0, 5, 10, 15, 30, or 60 min, the gradual movement of NF-κB into the nucleus was observed, with the most pronounced effect occurring within 30 min.The translocation of NF-κB persisted for the entire 60 min assessment period (Figure 4B).
The pretreatment of TNF-α antagonist, U0126, BAY 11-7082, and Lj-EtOH reduced the TNF-α-stimulated translocation of NF-κB, excepting TSIIA (Figure 4C).A reporter plasmid assay containing a human NF-κB response element was further used to delineate the potential mechanism.The effect of Lj-EtOH treatment on the modulation of NF-κB promoter reporter activity was assessed in the presence of signaling inhibitors.The activity of the NF-κB promoter reporter was decreased following exposure to Lj-EtOH, TNF-α antagonist, U0126, and BAY 11-7082.There was no significant change in reporter activity in GES-1 cells treated with TSIIA upon TNF-α stimulation (Figure 4D).The results suggest that Lj-EtOH successfully diminishes the expression of COX-2 and MMP-9 induced by TNF-α in normal GES-1 cells through the TNFR/ERK1/2/c-Fos and NF-κB pathways.

The Antimetastatic Activity of Ethanol Extract from Lonicera japonica Thunb. Was Evaluated In Vitro
The role of COX-2 and MMP-9 during gastric inflammation and cell migration is wellestablished [36].The primary objective of this study was to determine the effect of Lj-EtOH on TNF-α-induced functional alterations within GES-1 cells specifically mediated by COX-2 and MMP-9.A comprehensive analysis was conducted to determine the migratory behavior of GES-1 cells after a 24 h treatment with TNF-α.Lj-EtOH, a TNF-α antagonist, U0126, TSIIA, BAY 11-7082, MMP9i, or NS-398 pretreatment resulted in a significant inhibition of TNF-α-induced cell migration, as shown in Figure 5. Thus, Lj-EtOH plays a role in reducing cell migration by inhibiting the expression of COX-2 and MMP-9 expression in GES-1 cells.reaching confluence and serum starvation for 24 h, GES-1 cells were pretreated with 1 µg/mL Lj-EtOH, 1 µM TNF-α antagonist, 1 µM U0126, 10 µM TSIIA, 10 µM BAY 11-7082 for 1 h, 5 µM MMP9i, or NS-398 for 1 h.To evaluate cellular migration, the cell monolayer was scratched using a blue pipette tip, followed by incubation with 30 ng/mL TNF-α for 24 h.Phase contrast images of the cells were acquired at 24 h.(B) The number of migrating cells was counted.The data are presented as the mean ± SEM of three independent experiments.(*** p < 0.001 vs. control cells at 0 h; # p < 0.05, ## p < 0.01, compared to TNF-α-stimulated cells).

Discussion
Lj belongs to the Caprifoliaceae family, also known as Jin Yin Hua, and is a commonly used traditional Chinese medicine, health supplements, cosmetics, and ornamental groundcover [1].Lj could be used for functional/health supplement applications due to its intestinal benefits [37].In addition to Lj, general natural products are also widely used in promoting human health through various ways, such as Rhizoma polygonati extract [38], Gentiana extract [39], camel milk [40], safranal [41], and so on.Numerous studies have identified over 140 component compounds, including essential oils, flavones, organic acids, iridoids, saponins, and inorganic elements.Lj and its constituents demonstrate diverse pharmacological effects including anti-inflammatory, antiviral, antibacterial (including against Helicobacter pylori), antioxidant, hepatoprotective, anticancer, immune-boosting functions, insecticidal, acaricidal, antipregnancy, antihyperlipidemic, antithrombotic, and anti-lipase activities [1,2,42].In the present study, Lj-EtOH was examined based on its documented anti-inflammatory and gastroprotective properties.The induction of gastric inflammation has an important role in the development of gastric epithelial injury and the development of gastric inflammation-related disorders.
Chang et al. [43] found that LJ Flos (LJF) has been traditionally consumed orally as a medicinal plant and health food in China for years.To elucidate the gastrointestinal metabolism of LJF, three distinct in vitro models were used, specifically gastric juice, intestinal juice, and human intestinal bacteria.The identification of prototype compounds within the water extraction of LJF (LJF-WE) was accomplished through rigorous qualitative and quantitative analysis methodologies.They evaluated the stability of eight bioactive composites (sweroside, secoxyloganin, isochlorogenic acid B, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C) in simulated gastric fluid, intestinal fluid, and human fecal bacteria.The results indicated that these compounds exhibited a higher degree of stability when subjected to gastric and intestinal fluids compared with the presence of fecal bacteria.
Bang et al. [31] examined the gastroprotective properties of BST-104, which is a waterbased extract derived from LJ.They sought to elucidate the underlying mechanisms through the use of murine gastritis models induced by HCl/ethanol and gastric ulcer induced by acetic acid.The test subjects were orally administered BST-104, chlorogenic acid, or rebamipide, the latter serving as a positive control.The results indicated that BST-104 and its primary compound, chlorogenic acid, exhibited gastroprotective properties through their antioxidant activities, which involved enhanced levels of catalase, SOD, and GSH, whereas MDA levels were reduced.In addition, BST-104 and chlorogenic acid suppressed the secretion of proinflammatory cytokines (PGE 2 , TNF-α, IL-6, and IL-1β) by significantly downregulating the expression of NF-κB.Tang et al. [37] observed that Lj exhibited inhibitory effects on multiple cytokines, such as TNF-α, IL-1β, IL-6, IFN-γ, IL-12, and IL-17, in a murine model of DSS-induced ulcerative colitis.Furthermore, in a mouse model of induced immunosuppression through cyclophosphamide exposure, polysaccharide extracts derived from Lj successfully restored IL-2, TNF-α, and IFN-γ levels in the serum.This suggests that Lj's polysaccharide extracts hold promise as immunomodulatory agents.Previous studies primarily concentrated on determining the regulatory impact of Lj on the well-being of animal intestines through the use of in vivo models, namely mice and rats, along with in vitro models, including HMC-1 cells and RAW 264.7 cells, whereas limited research has been conducted on other species and cell lines.
The precise mechanism by which Lj manifests its gastroprotective effectiveness against TNF-α-activated inflammation in gastric mucosal epithelial cells has yet to be elucidated.Gastric inflammation, which is caused by proinflammatory cytokines, such as TNF-α secreted by activated immune cells, is dependent on the activation of NF-κB and MMP-9 through NF-κB signaling [44].Potter et al. [17] observed that a marked upregulation of COX-2 occurs in neoplastic sites or GC with inflammation.Moreover, Helicobacter pylori infection stimulates the TLRs/MyD88 and COX-2/PGE 2 pathways, leading to the activation of NF-κB and subsequent induction of an inflammatory response in tumor tissues [18].
The mechanism of TNF-α-induced MMP-9 occurs via the MAPK pathway in different cell lineages in response to inflammatory mediators [45][46][47][48].Furthermore, our study revealed that H. zeylanica-E 2 inhibits the TNF-α-induced activation of the proinflammatory cPLA 2 /COX-2/PGE 2 pathway in GC cells [19].This suggests that the COX-2/PGE 2 system may serve as an important pathological mediator in gastric inflammation-related disorders.In addition, we demonstrated that quercetin exhibits anti-inflammatory effects by suppressing the expression of TNF-α-stimulated MMP-9 in normal GES-1 cells [22].In the study, we assessed the potential protective effect of Lj against damage induced by TNF-α on COX-2 and MMP-9 expression in normal GES-1 cells.The cells were stimulated by TNF-α, which caused a reduction in COX-2 and MMP-9 expression in GES-1 cells following treatment with Lj.The results indicate that Lj exhibits the capacity to uphold the structural soundness of the gastric mucosa and may represent a treatment for inflammation.Previous studies implicated the MAPK family, specifically MAPK (extracellular p38, JNK1/2, and ERK1/2), in the underlying pathogenic mechanisms of gastric inflammation and gastric ulcer [49].Our results are consistent with these observations.In the present study, we explored the metastatic and inflammatory effects of Lj in normal GES-1 cells.Lj-EtOH ameliorated COX-2/PGE 2 and MMP-9 damage activated by TNF-α in normal GES-1 cells, highlighting its potential role in immune modulation of TNF-α-mediated inflammation.To determine the interplay among TNFR, NF-κB, ERK1/2, c-Fos, COX-2, and MMP-9 upon the response provoked by TNF-α, we evaluated distinct inhibitors, which included a TNF-TNFR, NF-κB, ERK1/2, c-Fos, COX-2, and MMP-9 inhibitors.
The data from IF stain analysis revealed that stimulation of TNF-α resulted in the phosphorylation and translocation of NF-κB (p65) and the activities of NF-κB/p65-Luc and AP-1-Luc were mediated through pathways involving TNFR-ERK1/2-c-Fos and NF-κB.In addition, Lj caused a reduction in the expression of COX-2/PGE 2 and MMP-9 induced by TNF-α in GES-1 cells and modulated the ERK1/2-c-Fos and NF-κB signaling.There is a plausible hypothesis suggesting that Lj effectively inhibits the activity of upstream molecules, such as TNFR, leading to the downstream inhibition of key signaling components, including NF-κB, ERK1/2, and c-Fos.The transcription factor NF-κB has a significant role in the modulation of inflammatory responses, given its involvement in the activation of proinflammatory cytokines.Following stimulation with these cytokines, NF-κB activity is activated, resulting from the phosphorylation and subsequent degradation of IκB.This results in the translocation of active NF-κB to the nucleus and stimulates the transcription of proinflammatory genes, cytokines, chemokines, adhesive proteins, and proteinases [50].
In the present study, we observed that pretreatment with BAY 11-7082 (NF-κB inhibitor) and TSIIA (c-Fos inhibitor) resulted in a reduction of TNF-α-induced COX-2/PGE 2 and MMP-9 expression in GES-1 cells.COX-2/PGE 2 and MMP-9 have emerged as promising targets for various therapies, which are the subject of many current studies [51,52].Understanding the role of inflammatory mediators will be valuable for designing therapeutic strategies to treat gastric inflammatory diseases.There is the potential for mitigating gastric damage through the suppression of COX-2/PGE 2 and MMP-9 production.Because of the widespread use of nonsteroidal anti-inflammatory drugs (NSAIDs), there is a need to discover substitute compounds to manage inflammation.Lj and its extracts may represent a promising alternative as dietary supplements for ameliorating the initial perturbations associated with gastric inflammation.Additionally, our findings indicate that TNF-α significantly induced an inflammatory response in GES-1 cells.On the other hand, IL-β or IFN-γ induced no significant effect.Therefore, we use TNF-α to focus on the role in gastrointestinal inflammation.Moreover, GES-1 belongs to the normal human gastric mucosa epithelial cell line, which is significantly close to normal physiological reactions.However, gastric cancer cell lines (such as AGS, AZ521, or TSGH) are far away from normal GES-1 cells to study gastric inflammatory-relative response.
From the data of the cell viability assay (Figure 2B,C), it seems that there existed a minimal effect on the viability of GES-1 cells upon treatment with Lj at concentrations ≤10 mM for 24 h.However, it is important to note that the safety and efficacy of Lj have not been definitively established, as indicated by previous studies [53].The optimal dosage of Lj may differ based on an individual's age, state of health, and additional factors.Although adverse effects resulting from Lj supplementation are infrequent, current data have not suggested an optimal dose range for Lj.The safety of natural products cannot always be assured, and determining a safe and effective dose is necessary [54,55].However, an intravenous formulation containing Lj along with two other herbs has been safely administered to children for up to 7 days.When taken orally, Lj flower extracts are potentially safe for a duration of up to 8 weeks.Skin contact with Lj may cause allergic individuals to develop a rash [56].Lj may exert anticoagulant properties and could interact with medications that also have anticoagulant effects, such as aspirin, plavix, cataflam, heparin, and coumadin, thus increasing the risk of bruising and bleeding.There is limited information regarding the use of Lj during pregnancy and breastfeeding, so it is advisable to avoid its use during these periods.Lj may have anti-inflammatory properties, although further studies are necessary to fully understand its mechanisms of action [57].

Conclusions
We examined the effect of Lj-EtOH on the metastatic and inflammatory properties of normal GES-1 cells in vitro.Lj-EtOH exhibits a protective effect against TNF-α-induced damage in normal GES-1 cells.Lj-EtOH specifically targeted the COX-2/PGE 2 system and MMP-9 to ameliorate their detrimental effects.To elucidate the underlying mechanisms, we examined a series of inhibitors, including a TNF-α antagonist, U0126 (a selective inhibitor of ERK1/2), TSIIA (a specific inhibitor of c-Fos), and BAY 11-7082 (an inhibitor of NF-κB) and determined their effects on TNFR, ERK1/2, c-Fos, and NF-κB in response to TNF-α exposure.Lj-EtOH inhibits the TNF-α-induced expression of COX-2/PGE 2 and MMP-9 in GES-1 cells.This effect is achieved through the inhibition of the proinflammatory TNFR-ERK1/2-c-Fos and NF-κB signaling pathways.Our results suggest that Lj-EtOH has potential as a therapeutic agent and warrants further consideration for normal gastric mucosal cells against disease-induced damage (see Figure 6.To identify the potential compounds in Lj-EtOH, the high-performance liquid chromatography profile and major compound structures were identified, as shown in Supplemental Figure S1.In addition, Lj-EtOH may be suitable as a food supplement to prevent early pathological emergence linked to gastric inflammation.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cimb46070433/s1, Figure S1: The high-performance liquid chromatography profile and major compound structures.

Figure 1 .
Figure 1.Induction of COX-2 and MMP-9 expression by TNF-α through the TNF-α receptor in normal GES-1 cells.Normal GES-1 cells were stimulated with TNF-α at a concentration of 30 ng/mL
• C (H 2 O extract, Lj-H 2 O) or 50 • C (EtOH extract, Lj-EtOH), a static extraction time of 15 min, 1 extraction cycle, and a constant pressure of 1500 psi.The extracts were collected in glass vials and evaporated on a rotary evaporator.Finally, the Lj extracts were stored at −20