Chinese liquor extract enhances inflammation resistance in RAW 264.7 and reduces aging in Caenorhabditis elegans

Recent reports have indicated that the ingredients in Chinese liquor possess multiple bioactivities. The objective of this study was to evaluate the potential effects of Chinese liquor extract (CME) on the resistance to inflammation in mononuclear macrophages (RAW 264.7 cell line) and aging in Caenorhabditis elegans (C. elegans). The results showed that CME suppressed key lipopolysaccharide (LPS)-induced pro-inflammatory mediators, tumor necrosis factor-α, and nitric oxide in vitro. Furthermore, CME inhibited activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/AKT pathways in LPS-stimulated cells. Further studies also showed that CME improved stress resistance of nematodes under infection conditions. Moreover, CME increased the expression of immune-related genes, such as lys-7. Based on these results, our findings provide mechanistic insights about the protection provided by CME against LPS-induced inflammation in RAW 264.7 cells, namely, inhibition of MAPK and PI3K/AKT pathways, as well as its capability against Pseudomonas aeruginosa- and Staphylococcus aureus-induced aging in C. elegans.


Introduction
Components of the innate immunity include neutrophils and macrophages, and their role is to initiate an inammatory response, form phagocytes to kill pathogens, recruit natural killer cells, and facilitate the maturation and migration of dendritic cells that will initiate the adaptive immune response. 1 Inammation, which is accompanied by fatal damages, such as oxidative damage, may worsen numerous diseases. However, inammation is not typically a negative phenomenon: it is the response of the immune system to the invasion of viruses or bacteria and other pathogens. Emerging evidence suggests that inammations are related to immunosenescence and reciprocally, controlling the inammatory status may allow a better chance of controlling immunosenescence. 2 Furthermore, inammation is an important and necessary part of the normal host responses to pathogens, but the overproduction of inammatory molecules might also cause immune-related inammatory diseases and eventually death. Therefore, low responders involved in regulation of innate defense mechanisms might better control inammatory responses and immunosenescence. 3 Lipopolysaccharide (LPS), a pathogenic endotoxin that can induce inammatory responses and oxidative stress, is widely used for evaluating anti-inammatory efficacy. In particular, macrophages produce excess pro-inammatory cytokines, tumor necrosis factor-a (TNF-a), and nitric oxide (NO) when exposed to inammatory stimuli, such as LPS. 1 Accumulating research shows that LPS causes overproduction of pro-inammatory mediators and cytokines by activating nuclear factor-kB (NF-kB), which is associated with mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/AKT pathways. 2 In addition, inammatory stress increases the production of reactive oxygen species (ROS) and reduces antioxidant enzymes. 3,4 Caenorhabditis elegans (C. elegans) is a commonly used model for aging as it exhibits the properties of a short lifespan, simple anatomy, and easy feeding. 5 In addition, C. elegans share many highly conserved biochemical pathways with humans. 6 Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), two common pathogens in humans, are capable of causing a diverse range of diseases from supercial skin infections and so tissue abscesses to life-threatening infections such as sepsis, endocarditis, pneumonia, and toxic shock syndrome. 7 Moreover, bacterial infections shorten the lifespan of nematodes and cause immunosenescence. 8 Aging is a process accompanied by deterioration of tissues, organs, and organism as well as the debasement of immunity, adaptability, and anti-infection ability, and nally the termination of life. 9 There is a reciprocal relationship between aging and immune function degradation. Therefore, anti-aging treatments can improve immune function, while improving immune function can delay aging. 10 Previous reports have shown that intake of antioxidants, such as natural products and herbal formulations, could enhance anti-infection ability and extend the lifespan of C. elegans. 11 Chinese liquor is an alcoholic beverage obtained from grains by complex fermentation processes. Research studied the aroma compounds in Moutai liquor with comprehensive twodimensional gas chromatography/time-of-ight mass spectrometry, and the results showed that a total of 528 components were identied in a Moutai liquor sample, including organic acids, alcohols, esters, ketones, aldehydes, acetals, lactones, and nitrogen-containing and sulfur-containing compounds, in addition to alcohol and water. 4 Some studies have shown that Moutai liquor does not cause liver injury, but rather strengthen lipid peroxidation in the liver, induce the increase in metallothioneins (MT), inhibit the proliferation of hepatic stellate cell (HSC) and generation of collagen and enhance the effect of antioxidation owing to multiple bioactive substances present in it. 5 Chinese liquor has historically been regarded as the most important Chinese medicine by herbalist doctors. 12 Recent reports have indicated that some substances in Chinese liquor, such as geraniol, possess a variety of bioactivities such as anticancer, anti-inammatory, and antibacterial properties. 13 Chinese liquor has been consumed in China for thousands of years, and numerous studies have reported that some ingredients in Chinese liquor have antiseptic, anti-inammatory, and antioxidant effects; however, no previous study has focused on the anti-infection effect of Chinese liquor. As infection-related morbidity and mortality is closely associated with environmental stress in a given population, potential stress-resistance effects of CME in C. elegans were also investigated in this paper. Evaluating the protective effect of CME and its underlying mechanisms will broaden the understanding of Chinese liquor and provide a reference for healthy drinking.

Chinese liquor extract preparation
Chinese liquor extract (CME) samples were prepared by the following method. Initially, 50 mL Chinese liquor, 20 mL H 2 O, and 90 mL CHCl 3 were added to a 250 mL separating funnel. The CHCl 3 layer was evaporated at 0 C using a Turbovap Sample Concentrator. Approximately 0.39 mg of the residue remained and was taken as CME and stored at À20 C in the dark. Mentions of brand names do not imply any research contact with the liquor manufacturer nor are these mentions for advertising purposes.
Cell and nematode culture RAW 264.7 cells were maintained in Dulbecco's Modied Eagle's Medium containing 10% (v/v) fetal bovine serum, Lglutamine (2 mM), penicillin (100 U mL À1 ) and streptomycin (100 U mL À1 ) at 37 C in a humidied atmosphere containing 5% CO 2 ; culture medium was changed every 2 days. Cells in logarithmic growth phase were selected for experiments and divided into the following six experimental treatment groups: control, LPS, ethanol + LPS, Mou + LPS, CME + LPS, and Ger + LPS. Nematodes were cultured at 25 C on nematode growth medium (NGM) plates and seeded with Escherichia coli OP50. Lyosgeny broth and slow-killing medium were prepared according to previously reported methods. 14 Determination of intracellular ROS and NO in RAW 264.7 cells The cytotoxic activity of CME was detected using a previously described method, 15 while intracellular ROS production was detected with a DCFH-DA using another previously reported method. 16 Briey, RAW 264.7 cells were pre-treated with chemicals for 24 h, and then stimulated with LPS (100 mg mL À1 ). Aer incubation for 24 h, the cells were stained with 10 mM DCFH-DA at 37 C in the dark. Aer 0.5 h, the cells were washed with phosphate-buffered saline (PBS) twice, and then placed in an Ascent FL uorescence plate reader (37 C). Emission at 535 nm was measured aer excitation at 485 nm for 20 min. For NO concentration detection, cells were cultured using the same method before collecting cell supernatants. NO concentration was detected using a commercial NO ELISA kit (Beyotime, Shanghai, China) according to the manufacturer's instructions.

Western blot analysis
RAW 264.7 cells were incubated with chemicals [geraniol, CME, Chinese liquor (53%), ethanol (v : v/ethanol : H 2 O/53 : 47)] for 24 h prior, and then stimulated with LPS (100 mg mL À1 ) for 24 h. Aer incubation, the cells were collected, washed with PBS, lysed in 100 mL lysis buffer containing protease and phosphatase inhibitor cocktails, and centrifuged at 13 000Âg for 15 min. An immunoblotting assay was performed as previously reported using b-actin protein as a measurement of the amount of protein analyzed. 17

Nematode lifespan assays
Nematodes were maintained on NGM plates and seeded with Escherichia coli OP50 at 25 C to obtain L4-larvae nematodes, as previously reported. 18 Young adult worms were transferred to NGM plates containing FUDR (50 mM) to prevent the growth of progeny. This transfer day was designated as day 0. For the safety assessment, CME, geraniol, Chinese liquor and ethanol were added to NGM plates (100 mL per plate) at concentrations ranging from 0-0.40 mg mL À1 for 48 h before detecting effects on lethality, growth, and movement behavior using previously reported methods. 19 Anti-Pseudomonas aeruginosa and anti-Staphylococcus aureus infection assays Experiments were performed according to a previously reported method with some modications. 20 The minimal inhibitory concentration (MIC) of CME, geraniol, Chinese liquor and ethanol against P. aeruginosa and S. aureus was detected using a previously reported method. 21 Briey, young adult worms were transferred to new NGM plates containing FUDR (50 mM) to prevent the growth of progeny, and then pretreated for 48 h in the following groups: blank (Con); ethanol (V : V/ethanol : H 2 O/53 : 47) (Eth); Chinese liquor (53%) (Mou); CME (CME); and geraniol (Ger). Synchronized young adult nematodes were cultured in NGM plates with or without chemicals for 24, 48, or 72 h. Aer incubation, approximately 80 nematodes were randomly picked and placed into plates coated with P. aeruginosa or S. aureus, which were cultured at 25 C; this day was considered as day 0. Each day, the number of surviving nematodes was counted until all nematodes were dead. Triplicate plates were used for each group. Nematodes that showed no movement on touching gently were considered dead.

Real time-polymerase chain reaction (RT-PCR) assays
Nematodes were treated as described above. Aer pretreatment with CME for 48 h and infection with P. aeruginosa or S. aureus for 48 h, total RNA was isolated from about 1500 nematodes using Trizol reagent. 22 Primer sequences for RT-PCR are listed in Table 1 and results are reported as mean AE SD.

Results and discussion
Cytotoxicity of CME on RAW 264.7 cells Aer obtaining the CME, we analyzed it with GC-MS, and the results showed that the CME is mainly composed of esters, polyols, acids, aromatic compounds, furans, aldehydes and pyrazines. HPLC analysis showed that the concentration of geraniol in Chinese liquor is 1.107 AE 0.013 mg L À1 (p > 0.05).
In consideration of the fact that compounds may themselves elicit cytotoxicity, the effect of each chemical on RAW 264.7 cell viability was tested. The results showed that no chemical tested (maximum concentration 50.0 mg mL À1 ) had a signicant effect on RAW 264.7 cell viability. Accordingly, the anti-inammatory effects of CME on RAW 264.7 cells were detected at 50.0 mg mL À1 .
Effects of CME on pro-inammatory mediators NO, a representative pro-inammatory mediator, is regulated by inducible NO synthase (iNOS). 23 In addition, major pro-inammatory cytokines, such as TNF-a, are overexpressed in macrophages stimulated by LPS, where they contribute to the pathogenesis of various inammatory diseases. 24 Aer treatment with LPS, uncontrolled activation of RAW 264.7 cells can Paper cause injury via overproduction of the inammatory mediators such as NO and TNF-a, 25 which is consistent with the results presented in Fig. 1 (NO and TNF-a content in the control group was set as 1). These results indicated that LPS stimulation increased the NO concentration (6.85 AE 0.75) compared with the control group, but was signicantly reduced by CME (5.32 AE 0.51). In addition, TNF-a was signicantly reduced on CME pretreatment (208.3 AE 25.2) compared with that observed in the LPS group (328.9 AE 33.4). These results suggested that CME played a signicant role in modulating inammatory cytokine expression. Geraniol showed a similar effect to CME: it reduced the concentration of NO by 50.8% and inhibited TNF-a protein expression by 48.7% compared with that of the LPSadministered group. Compared with the LPS group, the relative NO concentrations and TNF-a protein expressions in Chinese liquor and ethanol groups increased, but showed no signicant difference.

CME reduced ROS levels
Inammation and oxidative stress are now recognized as two important factors contributing to the development of many diseases, and ROS plays crucial roles in both processes. 26 Indeed, oxidative stressors would cause serious damage to lipids, proteins, and DNA. 27,28 Intake of red wine can naturally interrupt ROS expression and inhibit the progression of inammation and oxidative stress. 29 As shown in Fig. 2

CME inhibits NF-kB and depresses IkB-a pathways
The NF-kB signaling pathway is important for inammatory responses. Activation of NF-kB requires phosphorylation of upstream IkB kinase (IKK), which contains two catalytic subunits, IKKa and IKKb. 30 Upon stimulation with LPS or pro-inammatory cytokines, IKK is phosphorylated and activated by TGF-b-activated kinase 1 (TAK1), resulting in further phosphorylation and depression of IkB in the ubiquitination pathway. Subsequently, NF-kB releases from the IkB/NF-kB dimer and translocates from the cytoplasm into the nucleus, inducing further pro-inammatory gene expression and inammatory responses. Therefore, inhibition of the NF-kB pathway may have a potential therapeutic effect. As such, the effect of CME on NF-kB pathway inhibition was investigated. As shown in Fig. 3 and 4, LPS stimulation markedly increased NF-kB p65 expression by 61.0% and reduced IkB-a expression by 42.7% compared with those in the control group. However, with CME pre-treatment, NF-kB p65 expression was markedly inhibited and IkB-a expression was upregulated. Compared with the LPS group, expression of NF-kB p65 was reduced by 11.8% and that of IkB-a was increased by 35.1%. These results suggest that CME may act as a negative regulator of NF-kB activation. However, ethanol and Chinese liquor showed contrasting effects as they increased NF-kB p65 expression and reduced IkB-a expression compared with those in the LPS group. We inferred that the ethanol in these groups played the main functions.

CME inhibits activation of MAPK and PI3K/AKT pathways
MAPKs play signicant roles against inammation, and extracellular signal-regulated protein kinase (ERK) can regulate, at least in part, NO production, iNOS expression, and TNFa secretion. 31 Moreover, rapid and lasting activation of c-Jun NH(2)-terminal kinase MAPK (JNK) can be induced by LPS in RAW 264.7 cells, and inhibition of JNK reduces LPS-induced TNF-a activity. A previous study has also shown that the PI3K/ AKT signaling pathway is involved in inammation. 32 As shown in Fig. 3 and 4, LPS stimulation promoted the phosphorylation of JNK, p38 MAPK, ERK, as well as PI3K and AKT by approximately 92.5%, 92.1%, 71.3%, 57.1%, and 174%, respectively, compared with those in the control group. Moreover, LPS inhibited the expression of JNK, p38 MAPK, ERK, PI3K, and AKT. Furthermore, with CME treatment, JNK, p38 MAPK, ERK, PI3K, and AKT expression increased, and their phosphorylation was inhibited compared with LPS groups. Thus, we concluded that CME may suppress the inammatory response by inhibiting MAPK and PI3K/AKT pathways. However, both ethanol and Chinese liquor showed contrasting effects to CME, and played pro-inammatory roles in response to LPS. Effect of CME treatment on anti-aging in C. elegans. The young adult nematodes were treated with CME for 24, 48 and 72 h, then exposed to Pseudomonas aeruginosa (A) and Staphylococcus aureus (B) and monitored the survivals every hour (N $ 80 animals). The experiment was repeated multiple times and a representative trial is shown. Bars with different letters indicate significant difference between groups (p < 0.05).

CME enhanced the anti-infection ability of nematodes
C. elegans is a popular animal model for aging studies because it has a short lifespan, rapid generation, and experimental exibility. In addition, C. elegans shares highly conserved biochemical pathways and many similar aging aspects with humans. From the initial identication of single gene mutations capable of increasing aging to the insulin-like growth factor signaling pathway, molecular mechanisms of aging have gradually become an increased focus of research. C. elegans has become the major model organism contributing to the discovery of molecular mechanisms of aging. As previously reported, antioxidants increase the lifespan of nematodes, and some non-ethanol ingredients in Chinese liquor, such as geraniol, exhibit good antioxidant activity in vitro. 33 Resveratrol, a polyphenol antioxidant found in red wine, has been the subject of intense interest in recent years for a range of unique anti-aging properties. 34 Blueberry wine with proanthocyanidin also exhibited optimal anti-aging effects on C. elegans. 35 Clinically, alcohol is used for sterilization. In general, the substances in Chinese liquor may inhibit bacterial growth, thus affecting the lifespan of nematodes. Therefore, we tested the MIC of CME, geraniol, Chinese liquor and ethanol, to avoid this hypothesis. We found that treatment with 0-0.40 mg mL À1 of CME, geraniol, Chinese liquor and ethanol did not affect the growth of bacteria or development, brood size, movement behavior, or viability of nematodes. Thus, a concentration of 0.20 mg mL À1 CME, geraniol, Chinese liquor and ethanol were selected for subsequent studies.
Aer pre-treatment with CME for 24, 48, or 72 h, lifespans were greatly improved, as shown in Fig. 5. Mean lifespans in control groups were 44.6 AE 1.40 h and 39.5 AE 1.60 h aer infection with P. aeruginosa or S. aureus, respectively. However, with CME pre-treatment, mean lifespans in infected groups increased. With CME pre-treatment for 24, 48, or 72 h, mean lifespans were 47.4 AE 3.60 h, 52.3 AE 2.80 h, and 61.6 AE 6.00 h in P. aeruginosa-infected groups, respectively; compared with the control group, the mean lifespans increased by 6.37%, 17.4%, and 38.2%, respectively. In addition, the mean lifespans of nematodes in the S. aureus-infected groups were also enhanced to 43.6 AE 2.50 h, 48.2 AE 1.90 h, and 52.7 AE 4.90 h with CME pretreatment for 24, 48, and 72 h respectively; compared with the control group, the lifespans increased by 10.5%, 22.0%, and 33.5%, respectively. For the ethanol and Chinese liquor groups, the lifespans of nematodes did not extend, but instead shortened. We inferred that the presence of ethanol may play a harmful effect and weakened the anti-stress ability of nematodes.

Genetic requirements for increased anti-infection with CME treatment
Bacteria are capable of causing disease and infection, which can abbreviate the lifespan of nematodes and cause aging. Expression levels of immune and aging effector genes signicantly changed upon bacterial infection. During the initial stage of infection, C-Type lectin genes (e.g., clec-60) appear to be responsible for enhanced adhesion and antibacterial activity. 36 The lysozyme gene lys-7, a presumptive antimicrobial gene critical for the induction of immune response, is upregulated in response to bacterial infection. Regulation of lys-7 mRNA against bacterial infection gradually increases with time of exposure and upregulation. 37 The F08G5.6 gene belongs to a family of CUB-like proteins. Previous reports support the role Fig. 6 The relative expression level of mRNA including: lys-7, clec-60, F08G5.6 in C. elegans. Assay results were confirmed by RT-PCR, and the relative expression levels of the mRNA were determined. Bars with different letters indicate significant difference between groups (p < 0.05).
for proteins carrying CUB-like domains in immunity against pathogens in C. elegans. The F08G5.6 gene is regulated by daf-2/ daf-16 and p38 MAPK pathways. 38 Aer pre-treatment with CME for 48 h, nematodes were infected with P. aeruginosa or S. aureus for 48 h. Expression levels of immune-related genes were then tested by RT-PCR. The results are shown in Fig. 6. For the P. aeruginosa infection condition, relative expression of lys-7 and clec-60 were upregulated 1.56-and 2.36-fold, respectively, in the CME treatment group compared with that in the control group. However, F08G5.6 expression was not different from the CME treatment group (1.05-fold).
For S. aureus infection conditions, relative expression of lys-7 was upregulated (2.00-fold) in the CME treatment group compared with that in the control group. However, expression of F08G5.6 and clec-60 were not different in the CME treatment groups (1.10-and 0.99-fold). Notably, geraniol showed better protective effect than CME, but both ethanol and Chinese liquor showed contrary effects, where the relative expression of immune-related genes lys-7, clec-60, and F08G5.6 was downregulated compared with that of the control group. Expression of immune genes is closely related to the anti-infection ability of C. elegans. The present study showed that CME upregulated the relative expression of lys-7 during P. aeruginosa and S. aureus infections. Thus, we inferred that the immune gene lys-7 played a role in stress resistance against bacterial infection. However, according to the results listed in Fig. 6, the relative expressions of lys-7, clec-60, and F08G5.6 in the uninfected control groups (UCG) were higher than those in the CME and geraniol groups. We inferred that treatments can attenuate but not offset the harm of bacteria.

Conclusions
Chinese liquor, one of the famous distilled liquors, is consumed worldwide and has components with diverse bioactivities. CME suppressed LPS-induced production of the key pro-inammatory mediators NO and TNF-a in vitro. In addition, the MAPK/PI3K/AKT signaling pathway was inhibited by CME treatment in LPS-induced RAW 264.7 cells. Moreover, CME upregulated the expression of immune-related genes and enhanced the anti-infection ability of cells. Future investigations will aim to provide better understanding of the function of Chinese liquor. Indeed, both animal and human studies must be designed, as extrapolations cannot be made from cell culture studies to humans.

Conflicts of interest
The authors declare no conict of interest.