Callicarpa Nudiora Extract Exerts Anti-Inammatory Effect on H. Pylori-Associated Gastritis Through Repression of ROS/NLRP3/Caspase-1/IL-1 β Signaling Axis

Helicobacter pylori ( H. pylori ) is a major pathogenic factor for the development of gastric diseases including chronic gastritis and gastric cancer. Callicarpa nudiflora (CN), an air-dried leaves extract of Callicarpa nudiflora Hook. & Arn., has been found to exhibit a broad-spectrum antibacterial effect. In our study, we extracted the active ingredient from air-dried leaves of Callicarpa nudiflora, detected the effect of CN against H. pylori -infected GES-1 cells in vitro , and elucidated the underlying mechanism. GES-1 cells were cocultured with HPSS1 at MOI = 100:1 and treated with different concentrations of CN. Results indicated that CN not only significantly decreased cellular lactate dehydrogenase leakage, but also markedly attenuated H. pylori -induced cell apoptosis and ROS production in GSE-1 cells, therefore protecting gastric epithelial cells against injuries caused by H. pylori . CN also inhibited the secretions of inflammatory factors, such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6 and IL-8. Furthermore, CN remarkably decreased the expression levels of NLRP3, PYCARD, active Caspase-1. In conclusion, CN exhibited highly efficient protective effect against H. pylori -induced gastritis and cell damage; Mechanismly, CN suppressed H. pylori -triggered inflammatory response and pyroptosis through depressing ROS production and NLRP3 inflammasome activation via ROS/NLRP3/IL-1β signaling axis. Affinity States), NLRP3 Affinity Bioscience, United States). Antibody against GAPDH (5174S, Cell signaling technology, United States), Antibody against PARP1 (66520-1-AP, Proteintech, China), Antibody against Caspase-1 (SC-56036, Santa Cruz, United States), Antibody against Caspase-3 (9662S, Cell signaling technology, United States).


Introduction
Helicobacter pylori, a gram-negative spiral bacterium, infects almost half population of the world at one time or another [1]. In most cases, patients infected with H. pylori have no symptoms. Only~30% of the infected patients will progress to clinical symptoms [2]. H. pylori colonizes human gastric epithelial cells, which causes gastric mucosal layer edema and neutrophil infiltration, leads to chronic gastritis, atrophic gastritis, even develop to gastric cancer [3][4][5]. H. pylori is not only cause of more than 70% gastritis [6][7][8], but also the main carcinogen of gastric cancer by World Health Organization [9]. H. pylori was first isolated and described in 1983 by Barry Warren and Robin Marshall, who won the 2005 Nobel Prize in physiology or medicine "for their discovery of the bacterium H. pylori and its role in gastritis and peptic ulcer disease" [10]. H. pylori can express multiple pathogenic virulent factors, one of which is cytotoxin-associated gene A (CagA). CagA activates the expression of the nuclear factor (NF)-κB of gastric epithelial cells and then triggers proinflammatory cytokines expression [11]. H. pylori can also induce the release of pro-inflammatory factors and the associated oxidative damage. For instance, pro-inflammatory factors IL-8 could activate and aggregate neutrophils, thereby inducing ROS production [12]. Excessive ROS could activate NLRP3 inflammasome, which triggers the cleavage of pro-IL-1β, then transforming into activated IL-1β [13,14]. was filtered and the residue was boiled for another 1hour with 1.5 liters water. The collected decoction was combined, then freeze-dried (Eyela FDU-2110, Eyela Corp, Tokyo, Japan) to gain Callicarpa nudiflora powder (268 g). Next, we identification of chemical constituents of CN by UPLC-ESI-Q-TOF-MS (Table 1). Additionally, we analyzed four main compounds of CN by UPLC spectrum, of which acteoside accounts for 33.26%, forsythoside B 0.98%, 5,4'-dihydroxy-3,7,3'-trimethoxyflavone 0.42%, luteolin 0.33% (Fig. 2). In the in vitro study, Callicarpa nudiflora was dissolved in DMSO, and DMSO was used for control (the volume of DMSO < 0.5% in all experiments).

CN showed anti-HPSS1 activity in vitro
Because of the broad-spectrum antibacterial effect of CN, we first detected the inhibitory effect of CN on HPSS1, and the minimal inhibitory concentration (MIC) test showed that the MIC value of CN on HPSS1 was 2.5 mg/mL (Fig. 3A).

Effects of CN on GES-1 cells
In order to figure out toxic and negative effects of CN on gastric mucosa epithelial cells GES-1, we conducted CCK-8 assay with increasing CN concentrations. Results showed that CN presented toxic effects at 45 µg/mL, moreover higher concentration caused stronger toxicity (Fig. 3B).

CN had therapeutic effect rather than protective effect
In order to further explore the role of CN in GES-1 infected by HPSS1, we first explored the complex number of infections between HPSS1 and GES-1. According to the experimental results, we selected MOI of 100 for the subsequent experimental study (Fig. 3C). On this basis, we further studied the effect of CN before and after HPSS1 infection, and the results showed that the effect of CN after HPSS1 infection was better (Fig. 3D). Therefore, we chose to use CN for the treatment of HPSS1 infected GES-1 cells in the following studies.

CN counteracted the damage of H. pylori to GES-1 cells
To research the effects of CN on cell physiology and survival after incubation with H. pylori, the morphology, viability and the percentage of LDH leakage of HP-infected GES-1 cells was analyzed. In vitro, GES-1 cells of control group were in good condition, the cell morphology of model group deteriorated and the number decreased, CN mitigated the situation ( Fig. 4A ) . The cell viability of HP infection decreased significantly, and CN treatment increased the cell viability in a concentration dependent manner (Fig. 4B). The damage of HP infection on cells was also manifested in the release of LDH, The release of LDH in HP-infected cells increased significantly, and the release of LDH in CN group decreased gradually with the increase of concentration (Fig. 4C).

CN reduced the apoptosis of H. pylori-infected GES-1 cells
The apoptosis of H. pylori-infected GES-1 cell with or without CN was assessed by flow cytometry with Annexin V/PI double staining. Data manifested that HP infection caused 21.73% apoptosis in GSE-1 cells. However, CN treatment prevent GSE-1 cells from apoptosis in a concentration-dependent manner ( Fig. 5A & B). To further clarify the anti-apoptotic mechanism ， we detected apoptosis-related proteins by western blotting.
Results showed that H. pylori infection accumulated the cleaved PARP (89 kDa) and

CN alleviated inflammatory response of H. pylori-infected cells
The effects of CN on the production of cytokines (IL-1β, IL-6, IL-8 and TNF-α) in H.

Callicarpa nudiflora Hook. & Arn. is a genus of Callicarpa linn. of the family
Verbenaceae, and it is widely distributed in South China [24]. We have analyzed its main chemical constituents and pharmacological activity in our previous work [25]. Here, we demonstrated the anti-inflammatory effect of CN in H. pylori-associated gastritis (HAG), further, we found that CN exerted anti-inflammatory response through depressing H. pylori-induced ROS/NLRP3/IL-1β signaling axis activation.
There are some publications about pharmaceutical activity of CN, such as mosquito larvicidal activities [33], hepatoprotective effects [34], wound healing effects [35], dispersing edema and hemostasis [36], but there is no report on whether CN can protect cells from undergoing apoptosis. It's fact that H. pylori infection cause immune cells and epithelial cells to undergo apoptosis [37,38]. In the present study, we employed flow cytometry and western blotting, finding that CN reversed H. pylori-caused cell apoptosis in GSE-1 cells in a concentration-dependent manner. This clues us that CN can protect cells from apoptosis during the bacteria infection. About this point, CN could be developed a valuable clinic ancillary drug to counteract side effects of front-line chemical drugs in the future.
Functional experiments showed that CN reduced ROS production triggered by H. pylori infection in GSE-1 cells. What is more, we profound explored its anti-inflammatory mechanism by measuring the expression level of inflammatory factors IL-1β, IL-6, IL-8, TNF-α through ELISA test and detecting pivotal proteins level of ROS/NLRP3/IL-1β signaling through western blotting. All these evidences suggested that CN had anti-inflammatory effect in H. pylori-associated gastritis cell model. NLRP3 inflammasome, nucleotide-binding domain and Leucine-rich repeat containing receptors and the pyrin and HIN domain containing 3, belongs to the NLR protein family, which contains 22 members in human. The NLRP3 inflammasome was initially found to be activated by ATP and toxins [39]. Subsequently, a wide series of stimuli of damage-associated molecular patterns was identified, such as microbial, RNA viruses, ROS, excess glucose, amyloids, urate and cholesterol crystals [40][41][42][43][44][45][46][47]. PYCARD, also named ASC/TMS1, is a bipartite protein that consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD) domain motif. Once activated, NLRP3 recruits PYCARD to form inflammasome, where PYCARD interacts with the CARD of procaspase-1 and converts it to active casprocase-1, which converts the cytokine precursors pro-IL-1β and pro-IL-18 into the mature IL-1β and IL-18, eventually it causes pyroptosis [48]. Thus, nuclear PYCARD polymerization or oligomeritzation are considered to be critical mechanisms of NLRP3 inflammasome activation [49,50].

Conclusions
In our study, CN reversed H. pylori-induced ROS and NLRP3 increase in GES-1 cells, as a result, NLRP3 cannot recruit adequate PYCARD into nuclear to form ASC polymerization, speck formation and inflammasome, which inhibited H. pylori-induced procaspase-1 activation, in turn, prevent the maturation of IL-1β. To be summarized, CN suppressed H. pylori-triggered inflammatory response and pyroptosis through depressing ROS production and NLRP3 inflammasome activation via ROS/NLRP3/IL-1β signaling axis.

Materials and Chemicals
Campylobacter

H. pylori Strains and Growth Condition
HPSS1 was provided in a frozen state by National Centers for Disease Control, and maintained on blood agar plates and brain heart infusion broth supplemented with 5% Sterile defibrated sheep blood at 37°C under 10% CO2, 5% O2 and 85% N2.

Determination of MIC
The inhibitory effect of Callicarpa nudiflora extract (CN) (0.0025~40 mg/mL) on HPSS1 was evaluated by determination of minimal inhibitory concentration (MIC) according to the Chinese Pharmacopoeia 2010.

Evaluation of GES-1 Cell Viability After Incubation With H. pylori
The cell viability of GES-1 cell exposed to H. pylori and CN was evaluated by CCK-8 assay and percentage of LDH leakage.

CCK-8 Assay
GES-1 cells which were in logarithmic growth phase with good growth state, and were inoculated into 96-well plate with 5000 cells. Add 100 μL of culture medium to each well, and place them in an incubator of 5% CO2 at 37°C for overnight cultivation. Then the cells were treated with ascending concentration of CN according to groups. After the time required for cell culture, PBS was used to rinse 4 times, and 100 μL fresh medium containing 10 μL CCK-8 reagent was added to each well, keep culturing at 37°C for 4 hours. The absorbance OD450 was determined by multifunctional microplate reader (Flexstation 3, Molecular Devices, United States).

LDH Measurement
LDH assay was used to quantity LDH release from cells. According to the directions, we collected the cell culture supernatant, centrifuged at 3000 rpm for 10 minutes, and measured the OD of the supernatant directly. The LDH concentration in the medium was measured at 440 nm. LDH activity (U/L) = (determination of ODcontrol OD)/(standard ODblank OD) * concentration of standard (2 mmol/L) *1000.

Apoptosis Detection
GES-1 cells in the logarithmic growth phase with good growth state were inoculated into 6-well plates with 10 5 /well. 2 mL of culture medium was added to each well and cultured overnight in a 5% CO2 incubator at 37°C. Then the cells were processed in according to groups. The cell pellets were collected, washed in PBS, resuspended cells with 500 μL binding buffer, 5 μL AnnexinV-FITC and 5 μL PI was added and mixed. At room temperature, the reaction was kept out of light for 5~15 minutes. To be detected by Flow cytometry (Beckman coulter, cytoFLEX, United States).

ROS Measurement
GES-1 cells in the logarithmic growth phase with good growth state were inoculated into 6-well plates with 10 5 /well. 2 mL of culture medium was added to each well and cultured overnight in a 5% CO2 incubator at 37°C. Then the cells were processed according to groups.
The cell pellets were collected, washed in PBS, added 1 mL DCFH-DA which was diluted with serum-free medium to 10 M, Incubated at 37°C for 20 minutes, and mixed once every 3 minutes. Then the cells were washed twice with serum-free medium, resuspended with PBS, to be tested by Flow cytometry (Beckman coulter, cytoFLEX, United States).

ELISA assay for IL-6, IL-8, IL-1β and TNF-α
Supernatants from GES-1 cell cultures were collected and centrifuged to remove cell debris.
The concentration of IL-6, TNF-α, IL-1β and IL-8 in the culture supernatants was determined by using a cytokine specific ELISA kit per the manufacturer's instructions. All assays were performed in triplicate in three independent experiments (FlexStation® 3, Molecular Devices, United States).

Western Blot
Total proteins were extracted with RIPA lysis buffer (Beyotime) with phosphatase and protease inhibitors, and the protein concentrations were detected with BCA Protein Assay Kit (Beyotime). SDS-PAGE was used to separate lysates, and then the proteins were transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 hours at room temperature, and incubated with primary antibody overnight at 4°C. After being incubated with secondary antibody, the signals were detected with ECL regents. Antibodies used were: GAPDH, PYCARD, PARP1, Caspase-1, NLRP3, Caspase-3.

Statistical Analysis
The data was analyzed with GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA).
Parametric tests were used to compare different treatments with Unpaired t-test. The data are expressed as the mean ± standard error, and the differences were considered significant as indicated as follows: *p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.001; ns: not significant.  Figure 1 The plant specimen picture of Callicarpa nudi ora.