Studies on Mechanism of Preventive Effect of Fermentation Product of Calvatia lilacina on Water-Immersion Stress Induced Gastric Ulcers in Mice

De Hu, Jia Wang, Huan Wang, Xiang-Yang Leng, Tian-Yi Zhao* and

Shu-Min Wang*

College of Pharmacy, Changchun University of Chinese Medicine, Changchun 130117, PR China.

ABSTRACT

To explore the potential mechanism of the fermentation product (FP) of Calvatia lilacina on gastric ulcer, fifty mice were randomly divided into 5 groups (10 mice per group), namely control (normal saline), model (normal saline), ranitidine group (0.03 g/kg) and FP groups (1 and 2 g/kg). Stress gastric ulcer was induced by water-immersion and restraint stress (WIRS) after 10 days of drug administration. To observe the histopathological changes of gastric mucosa. the activities of pepsin, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) in gastric tissue and the levels of tumor necrosis factor (TNF–α) and interleukin (IL-6) in serum were measured through ELISA, and the contents of NLRP3, ASC, caspase-1 and IL-1β in gastric tissue were detected by Western blot. The results showed that FP of C. lilacina could effectively prevent the stress gastric ulcer induced by WIRS in mice by reducing the expression level of NLRP3/ASC/caspase-1.

Article Information

Received 23 February 2021

Revised 28 May 2021

Accepted 05 November 2021

Available online 22 December 2021

(early access)

Published 30 May 2022

Authors’ Contribution

DH conceived the study, designed the experiments, supervised the research, analyzed the data and revised the manuscript. HW and JW completed the experiments and analyzed the data. DH wrote the manuscript. TYZ, XYL and SMW did the experiments.

Key words

Calvatia lilacina, Fermentation product, Gastric ulcer, Inflammation, Water-immersion, Restraint stress

DOI: https://dx.doi.org/10.17582/journal.pjz/20210223080239

* Corresponding author: [email protected]; [email protected]

0030-9923/2022/0005-2085 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Gastric ulcer is a common disease, the incidence of which is increasing year by year (Hung et al., 2019; Zakaria et al., 2011). There are many causes of gastric ulcer, including irregular diet, long-term drinking, the use of non steroidal anti-inflammatory drugs (NSAIDs), helicobacter pylori infection, work pressure, mental anxiety, etc., (Hunt et al., 2015). In recent years, with the acceleration of the pace of human life and the adjustment of dietary structure, negative emotions and life pressure have gradually become the main causes of gastric ulcers (Lee et al., 2017).

The occurrence of stress gastric ulcer is mainly acute gastric mucosal lesion characterized by inflammatory erosion, superficial ulcers and bleeding under stress condition, including various trauma, critical illness, shock, etc. (Cheng et al., 2017). In the stress state, the protective barrier of gastric mucosa was destroyed, and the increase of endogenous and exogenous attack factors accelerated the apoptosis of gastric mucosa cells, resulting in the disorder of gastric mucosa cell metabolism, the disorder of blood circulation of gastric mucosa, and the destruction of mucus mucosa barrier (Kubben et al., 2007). At present, the mechanism of stress gastric ulcer is not clear. Studies have shown that the main causes of stress gastric ulcer may be the decrease of pH value of gastric juice, mucosal ischemia, oxidative stress and over expression of inflammatory factors (Jo et al., 2013; Ye et al., 2013; Sindhu and Kuttan, 2012).

Puffballs are large fungus, mainly grown on the grassland in the wild. Edible when young, fruiting body can be used medicinally after maturity and have a variety of pharmacological effects, including bacteriostasis, anti-inflammatory, antitussive, antitumor and cell proliferation inhibition (Lee et al., 2018; Eroğlu et al., 2016; Tsay et al., 2009).

Therefore, the liquid fermentation products of C. lilacina was selected as a material in this experiment. Through this study, a stable, reliable, fast and high-yield fermentation technology of C. lilacina was determined, and a large number of fermentation products were obtained and the effect of fermentation produced by C. lilacina was further investigated.

MATERIALS AND METHODS

Materials and reagents

Calvatia lilacina (Mont. et Berk.) Lloyd was provided by the Mycological Medicine Research Laboratory of Jilin Bioengineering Technology Center. The ELISA assay kits for IL (interleukin)-6, TNF-α (tumor necrosis factor α), Pepsin, SOD (superoxide dismutase), MDA (malondialdehyde), and NO (nitric oxide) were purchased from Meimian Biotechnology (Yancheng, Jiangsu, China). Mouse anti-NLRP3, ASC, caspase-1, IL-1β and GAPDH were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). All other reagents used in the experiment are analytically pure. All other reagents used in the experiment were analytically pure. PMSF and RIPA were obtained from Beyotime Biotechnology (Shanghai, China).

Preparation of fermentation products of C. lilacina

The fermentation was cultured in a liquid shake flask, 120 mL of liquid medium (maltose 4%, yeast powder 3%, KH2PO4 0.1%, MgSO4 0.05%) was put into a 500 mL conical flask, inoculated according to 10% volume ratio (V/V), the shake flask speed was 150r/min, at 25°C for 7 days. When the biomass reached 1.25g/100 mL, the fermentation broth was collected, concentrated, dried and pulverized as experimental drugs.

Animals and experimental groups

Total 50 healthy male BALB/c mice (an albino and laboratory-bred strain, weighing 19-21g) were provided by Liaoning Changsheng Biotechnology Co., Ltd., (Liaoning, China; Certificate No.: SCXK Liao 2015-0001). Before the experiment, the mice were allowed to adapt to the standard laboratory conditions for a week, with free diet and drinking water, and were randomly divided into 5 groups (n=10): the control group (physiological saline, healthy mice), model group (physiological saline, model mice), ranitidine group (0.03g/kg), and fermentation product (FP) groups (1 and 2g/kg, respectively). Each group was given corresponding drugs by intragastric administration, respectively, for ten consecutive days.

Water-immersion stress induced gastric ulcers in mice

Before modelling, all mice were fasted for 24 h with sufficient drinking water supplied. In addition to control group, mice in other groups were fixed on the self-made mouse board, immersed in water at 20±1°C for 20 h, and the water surface was kept at the same level as the xiphoid process of mice (Rahmadi, 2021). Subsequently, for all groups of mice, blood was collected from eyeballs and sacrificed by cervical dislocations. The stomachs of three mice in each group were ligated at the pylorus, injected with an appropriate amount of 4% paraformaldehyde solution, then ligated at the cardia, and finally the free gastric tissues were immersed in 4% paraformaldehyde solution for fixation. The stomachs of the other mice were opened along the great curvature of the stomach and the stomach contents were removed with cold normal saline and the stomach tissue was frozen immediately.

Histopathological examination

A part of stomach tissue fixed in 4% paraformaldehyde solution and was embedded in paraffin. Tissue slices were cut at 5μm thickness and stained with hematoxylin and eosin. The histopathological changes of the stomach were observed under the light microscope.

Determination of related biochemical indices in gastric tissues

The stomach tissues were weighed, and cold saline (1:9) was added according to the mass ratio of organ mass and fully grinded to get 10% tissue homogenate. The tissue homogenates were centrifuged at 3500 rpm at 4°C for 10 min. Supernatant was used for estimation of the activities of pepsin, SOD, MDA and the content of NO.

Determination of inflammatory factors in serum

The blood samples were centrifuged at 3500 r/min, at 4°C for 10 min. The serum was used to determine the levels of TNF-α and IL-6 according to the instructions of ELISA kit. The standard curve was used to calculate the level of the analytes.

Western blotting analysis of gastric tissue

Briefly, 100 mg gastric tissue samples were homogenized in 500 μL precooled protein lysate (containing PMSF) and incubated on ice for 30 min. The homogenate was then centrifuged at 3500 rpm, 4°C for 10 min.

The supernatant was used for determination of the protein concentration with BCA kit. Equal amounts of total protein were subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose membranes. The nonspecific binding was prevented by incubation with 5% skimmed milk powder for 2 h at room temperature. Blots were incubated with primary antibody (anti-NLRP3, anti-Caspase-1, anti-ASC, anti-IL-1β, and anti-GAPDH) overnight at 4°C, and then, incubated the second antibody with fluorescent label for 2 h at room temperature. The bands were detected using the enhanced chemiluminescence detection system and band intensities were measured by densitometric analysis using Image J software. The ratio of NLRP3, Caspase-1, IL-1β or ASC to the corresponding control GAPDH was calculated for each sample.

Statistical analysis

The data were processed by Graph Pad Prism (Version 5.0) and the difference between groups was analyzed by one-way ANOVA. The experimental data were expressed as mean ± SEM, and P<0.05 suggested that the difference was statistically significant.

RESULTS

FP of C. lilacina could prevent gastric ulcer in mice induced by WIRS

The stress gastric ulcer was established in the restrained water soaked mice (Fig. 1). The results of histopathology of gastric mucosa showed that the gastric mucosa of the control group showed normal flesh color, mucosal folds were smooth, and no bleeding spots were seen. In the model group, diffuse bleeding spots were seen, gastric mucosa showed congestion, edema, bleeding and erosion spots, and inflammatory cell infiltration was obvious; The surface of the stomach tissue in the tidine group, high-dose enzyme group and low-dose enzyme group was smoother, with mild congestion and edema in part of the mucosa, a small amount of erosion, no obvious ulcers, and a small amount of inflammatory cell infiltration (Fig. 1).

 

Pepsin is a kind of proteolytic enzyme, which can decompose proteins and is one of the attack factors of gastric mucosa damage (Samuels and Johnston, 2020). Therefore, reducing pepsin activity is an important aspect of anti-gastric mucosal injury. Compared with the model group, the pepsin activity of ranitidine group and high-dose group of FP decreased significantly (P<0.05 or P<0.01) (Fig. 2).

 

FP of C. lilacina could affect the related indices of oxidative stress in gastric tissue of model mice

As shown in Figure 3, compared with the FP groups, the stage of MDA and NO in the gastric homogenate of the model group was elevated drastically (P <0.05 or P <0.01), and the degree of SOD in the gastric homogenate of the model group diminished extensively (P <0.01). Compared with the model group, the degree of MDA and NO in gastric homogenate of mice in ranitidine group, high-dose and low-dose of FP group decreased. The MDA of high dose and low dose of FP groups lowered substantially (P <0.05 or P <0.01). However, the degree of NO reduced barring vast difference. The degree of SOD in gastric homogenate of mice in ranitidine group and FP groups were first increased, though not significantly after that.

 

FP of C. lilacina could affect the anti-inflammatory indices in the serum of model mice

The degrees of TNF-α and IL-6 in the model group were drastically decreased (P <0.05 or P <0.01) compared with the FP groups, as shown in Figure 4. Compared with the model group, the ranges of TNF-alpha and IL-6 in serum of ranitidine group, the high-dose and low-dose group of FP were decreased, and the degrees of TNF-α and IL-6 in serum of high dose and low dose of FP groups were decreased appreciably (P<0.05 or P<0.01).

 

FP of C. lilacina can inhibit the activation of NLRP3/ASC/caspase-1 pathway in gastric tissue

Figure 5 shows that compared with the control group, the expression levels of NLRP3, caspase-1, ASC and IL-1β protein in the gastric tissues of the model group were significantly increased (P <0.01). The expression levels of NLRP3, caspase-1, ASC and IL-1β protein in the gastric tissues of the ranitidine group, the high-dose group of FP and the low-dose group of FP were significantly decreased (P <0.05 or P <0.01), compared with the model group.

 

DISCUSSION

Acute gastric ulcers are superficial diffuse gastric mucosal lesions, which are usually caused by excessive smoking and drinking, heavy use of non-steroidal anti-inflammatory drugs and severe stress injuries, such as shock, burn and surgical injury (Jeon et al., 2020; Rahman et al., 2020). Acute gastric ulcer can lead to serious upper gastrointestinal bleeds with high incidence and mortality rates (Li and Song, 2020). The mechanisms un-derlying acute gastric ulcers remain largely unclear, but acute inflammation can appear as the main factor that affects and regulates the progression of the disease.

Although there is evidence that the C. lilacina exerts a wide range of health-promoting effects (Zhu et al., 2010), its role in water-immersion stress induced acute gastric ulcers remains largely unknown. Therefore, this study was aimed to determine the potential effects of oral administration of the FP of C. lilacina on ethyl alcohol-induced acute gastric ulcers. As mentioned above, acute gastric ulcers were triggered in mice via water-immersion stress method, and the FP of C. lilacina exhibited protective effects on Water-immersion stress induced acute gastric ulcers. water-immersion stress did cause significant increase in MDA and NO, but a remarkable reduction in SOD. After treatment with the FP of C. lilacina, the levels of MDA and NO were significantly reduced, while SOD activity was markedly increased.

NLRP3 inflammasome activation and IL-1β release are considered to be one of the important mechanisms of diseases such as acute gastric injury. The binding of NLRP3 and ASC under different stimuli can trigger the release of caspase-1, resulting in pro-IL-1β activation and IL- 1β maturation (Zhou et al., 2020). NLRP3/ASC/caspase-1 pathway plays an essential role in water-immersion stress induced gastric injuries Higashimori et al. (2021). In this study, the expression levels of ASC, caspase-1, NLRP3 and IL-1β were examined to elucidate the antiinflammatory mechanisms of the FP of C. lilacina.

CONCLUSION

In summary, this study reveals that the fermentation product of Calvatia lilacina can significantly down-regulate the expression of NLRP3 and ASC, inhibit the activation of Caspase-1, reduce the release of IL-1β, TNF-α and IL-6, and effectively reduce Water-immersion stress induced inflammation, enhance the anti-oxidation activity of gastric mucosa, and thus reduce the severity of gastric ulcer. It is suggested that the FP of C. lilacina may confer protective roles in Water-immersion stress induced acute gastric ulcer by inhibiting NLRP3 signaling pathway.

ACKNOWLEDGEMENTS

This study was financial supported by the National Natural Science Foundation of China (Grant No. 81274037). We would like to express special thanks to Shuang-Shuang Mu for his assistance and contribution to this article.

Ethical compliance

All procedures and experimental methods were approved by the Animal Ethics Committee of Changchun University of Traditional Chinese Medicine (Certificate No.: 20180126).

Statement of conflicts of interest

The authors have declared no conflict of interest.

REFERENCES

Cheng, Y.T., Lu, C.C. and Yen, G.C., 2017. Phytochemicals enhance antioxidant enzyme expression to protect against NSAID-induced oxidative damage of the gastrointestinal mucosa. Mol. Nutr. Fd. Res., 61: 1600659. https://doi.org/10.1002/mnfr.201600659

Eroğlu, C., Seçme, M., Atmaca, M., Kaygusuz, O., Gezer, K., Bağcı, G. and Dodurga, Y., 2016. Extract of Calvatia gigantea inhibits proliferation of A549 human lung cancer cells. Cytotechnology, 68: 2075-2081. https://doi.org/10.1007/s10616-016-9947-4

Higashimori, A., Watanabe, T., Nadatani, Y., Nakata, A., Otani, K., Hosomi, S., Tanaka, F., Kamata, N., Taira, K., Nagami, Y., Tanigawa, T. and Fujiwara, Y., 2021. Role of nucleotide binding oligomerization domain-like receptor protein 3 inflammasome in stress-induced gastric injury. J. Gastroenterol. Hepatol., 36: 740-750. https://doi.org/10.1111/jgh.15257

Hung, K.W., Knotts, R.M., Faye, A.S., Pont, A.R., Lebwohl, B., Abrams, J.A. and Freedberg, D.E., 2019. Factors associated with adherence to Helicobacter pylori testing during hospitalization for bleeding peptic ulcer disease. Clin. Gastroenterol. Hepatol., 18: 1091-1098. https://doi.org/10.1016/j.cgh.2019.07.037

Hunt, R.H., Camilleri, M., Crowe, S.E., El-Omar, E.M., Fox, J.G., Kuipers, E.J., Malfertheiner, P., McColl, K.E., Pritchard, D.M. and Rugge, M., 2015. The stomach in health and disease. Gut, 64: 1650–1668. https://doi.org/10.1136/gutjnl-2014-307595

Jeon, D.B., Shin, H.G., Lee, B.W., Jeong, S.H., Kim, J.H., Ha, J.H., Park, J.Y., Kwon, H.J., Kim, W.J., Ryu, Y.B. and Lee, I.C., 2020. Effect of heat-killed Enterococcus faecalis EF-2001 on ethanol-induced acute gastric injury in mice: Protective effect of EF-2001 on acute gastric ulcer. Hum. exp. Toxicol., 39: 721-733. https://doi.org/10.1177/0960327119899987

Jo, I.G., Park, D., Kyung, J., Kim, D., Cai, J., Kim, J., Kwak, T.H., Yoo, S.K., Jeong, H.S. and Kim, Y.B. 2013. Inhibitory effects of a β-dunnione compound MB12662 on gastric secretion and ulcers. Lab. Anim. Res., 29: 178-181. https://doi.org/10.5625/lar.2013.29.3.178

Kubben, F.J., Sier, C.F., Schram, M.T., Witte, A.M., Veenendaal, R.A., van Duijn, W., Verheijen, J.H., Hanemaaijer, R., Lamers, C.B. and Verspaget, H.W., 2007. Eradication of Helicobacter pylori infection favourably affects altered gastric mucosal MMP-9 levels. Helicobacter, 12: 498–504. https://doi.org/10.1111/j.1523-5378.2007.00527.x

Lee, S., Lee, D., Lee, J.C., Kang, K.S., Ryoo, R., Park, H.J. and Kim, K.H., 2018. Bioactivity-guided isolation of antiinflammatory constituents of the rare mushroom, Calvatia nipponica, in LPS-stimulated RAW264.7 macrophages. Chem. Biodiver., 15: e1800203. https://doi.org/10.1002/cbdv.201800203

Lee, Y.B., Yu, J., Choi, H.H., Jeon, B.S., Kim, H.K., Kim, S.W., Kim, S.S., Park, Y.G. and Chae, H.S., 2017. The association between peptic ulcer diseases and mental health problems: A population-based study: A STROBE compliant article. Medicine, 96: e7828. https://doi.org/10.1097/MD.0000000000007828

Li, Y. and Song, Y., 2020. Diagnostic value of serum gastrin and epidermal growth factor to the gastric ulcer complicated with upper gastrointestinal hemorrhage. J. Coll. Phys. Surg. Pak., 30: 1269-1272. https://doi.org/10.29271/jcpsp.2020.12.1269

Rahmadi, M., Su’aida, N., Yustisari, P., Agung Dewaandika, W., Oktavia Hanaratri, E., Andarsari, M.R., Sumarno, Aryani T., 2021. Gastroprotective effect of fluvoxamine and ondansetron on stress-induced gastric ulcers in mice. J. Basic Clin. Physiol. Pharmacol., 32: 485-490. https://doi.org/10.1515/jbcpp-2020-0424

Rahman, Z., Dwivedi, D.K. and Jena, G.B., 2020. Ethanol-induced gastric ulcer in rats and intervention of tert-butylhydroquinone: Involvement of Nrf2/HO-1 signalling pathway. Hum. Exp. Toxicol., 39: 547-562. https://doi.org/10.1177/0960327119895559

Samuels, T.L. and Johnston, N., 2020. Pepsin in gastroesophageal and extraesophageal reflux: molecular pathophysiology and diagnostic utility. Curr. Opin. Otolaryngol. Head Neck Surg., 28: 401-409. https://doi.org/10.1097/MOO.0000000000000664

Sindhu, E.R. and Kuttan, R., 2012. Carotenoid lutein protects rats from gastric ulcer induced by ethanol. J. Basic clin. physiol. Pharmacol., 23: 33-37. https://doi.org/10.1515/jbcpp-2011-0032

Tsay, J.G., Chung, K.T., Yeh, C.H., Chen, W.L., Chen, C.H., Lin, M.H.C., Lu, F.J., Chiou, J.F. and Chen, C.H., 2009. Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells. J. Agric. Fd. Chem., 57: 1579-1588. https://doi.org/10.1021/jf8030265

Ye, B, Zhou, P.Y., Jia, M., Cheng, X.S., Jia, Y.T., and Xu, S.G., 2013. Absence of NF-κB subunit p50 ameliorates cold immobilization stress-induced gastric ulcers. Biochem biophys. Res. Commun., 434: 547-551. https://doi.org/10.1016/j.bbrc.2013.03.112

Zakaria, Z.A., Abdul-Hisam, E.E., Rofiee, M.S., Norhafizah, M., Somchit, M.N., Teh, L.K. and Salleh, M.Z., 2011. In vivo antiulcer activity of the aqueous extract of Bauhinia purpurea leaf. J. Ethnopharmacol., 137: 1047-1054. https://doi.org/10.1016/j.jep.2011.07.038

Zhou, Y., Zhang, C.Y., Duan, J.X., Li, Q., Yang, H.H., Sun, C.C., Zhang, J., Luo, X.Q. and Liu, S.K., 2020. Vasoactive intestinal peptide suppresses the NLRP3 inflammasome activation in lipopolysaccharide-induced acute lung injury mice and macrophages. Biomed. Pharmacother., 121: 109596. https://doi.org/10.1016/j.biopha.2019.109596

Zhu, Y., Yuan, S. and Li, B. 2010. Scavenging effect of water-soluble polysaccharide in purple bald mabo on oxygen free radical. J. Anhui agri. Sci., 38: 4053-4055 (in Chinese). https://doi.org/10.13989/j.cnki.0517-6611.2010.08.069