Tormentic acid protects alcohol induced GES-1 cells via regulating Keap-1/Nrf2 and NLRP3 inflammasome signaling pathways

doi:10.12092/j.issn.1009-2501.2026.08.004

Abstract

Abstract:

AIM: To investigate the protective effect and mechanisms of tormentic acid (TA) on GES-1 cells induced by alcohol. METHODS: The GES-1 cell injury model induced alcohol was established. The GES-1 cells were divided into control group, model group, TA (12.5 μg/mL) group, Nrf2 inhibitor group (ML385, 10 μmol/L) group and NLRP3 inhibitor (MCC950, 8 μmol/L) group. After adding the corresponding drugs to the drug intervention group cells and culturing them for 22 hours, cells were treated with 7% alcohol except for the control group and cultured for another 2 hours, and then the cell supernatant and cells were collected, respectively. MTT assay was used to test cell viability; Cell scratch and Transwell were utilized to detect cell migration; AnnexinV FITC/PI double staining was used to test cell apoptosis; Immunofluorescences were used to detect MMP and ROS level in alcohol induced GES-1 cells; Colorimetric and ELISA methods were used to test the levels of LDH, IL-4, IL-1β, IL-6, IL-10, IL-18, NO and TNF-α in the GES-1 cell supernatants; Colorimetric method was utilized to detect the levels of CAT, GSH, MDA, MPO, SOD, T-AOC in alcohol induced GES-1 cells, and cytochrome C in the cytosol and mitochondria of GES-1 cells; Immunofluorescence was utilized to detect the co-localization of NLRP3, ASC and caspase-1; The changes in intracellular ultrastructure were observed under an electron microscope; Real-time PCR was used to detect the mRNA expression levels of Apaf-1, ASC, Bcl-2, Bcl-xl, Bad, Bax, caspase-1, COX-1, COX-2, GCLC, HO-1, iNOS, Keap-1, NEK7, NLRP3, NQO1, Nrf2, PGE2 and TXNIP in alcohol induced GES-1 cells; Western blot was utilized to test the protein expression levels of Apaf-1, ASC, Bcl-2, Bcl-xl, Bad, Bax, caspase-1, cleaved-caspase-3, cleaved-caspase-9, PARP-1, cleaved-PARP-1, GCLC, HO-1, Keap-1, NEK7, NLRP3, NQO1, nuclear Nrf2, pro-caspase-1, pro-caspase-3, pro-caspase-9, pro-IL-18, pro-IL-1β, total Nrf2 and TXNIP in GES-1 cells. RESULTS: Compared with the model group, TA prominently promote alcohol induced GES-1 cell migration, depressed cell apoptosis and LDH release, substantially reduced intracellular ROS level, IL-1β, IL-6, IL-18, NO, TNF-α levels in the GES-1 cell supernatants, MDA, MPO levels in alcohol induced GES-1 cells and cytochrome C content in cytosol (P<0.01), substantially elevated mitochondrial membrane potential, IL-4, IL-10 levels in the GES-1 cell supernatants, CAT, GSH, SOD, T-AOC levels in alcohol induced GES-1 cells and cytochrome C content in the mitochondria (P<0.01). It dramatically down-regulated the ASC, Apaf-1, Bad, Bax, caspase-1, COX-2, iNOS, Keap-1, NEK7, NLRP3, TXNIP mRNA and ASC, Apaf-1, Bad, Bax, caspase-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP-1, Keap-1, NEK7, NLRP3, pro-caspase-1, pro-IL-1β, pro-IL-18, TXNIP protein expressions in alcohol induced GES-1 cells, restrained co-localization of NLRP3, ASC and caspase-1 in alcohol induced GES-1 cells; The structure of the nucleus and nucleolus was intact, chromatin was uniform, and mitochondrial swelling was reduced; TA up-regulated the COX-1, Bcl-2, Bcl-xl, GCLC, HO-1, NQO1, Nrf2, PGE2 mRNA and Bcl-2, Bcl-xl, GCLC, HO-1, NQO1, total Nrf2, nuclear Nrf2, pro-caspase-3, pro-caspase-9 protein expressions and Bcl-2/Bax, Bcl-xl/Bad ratios in the alcohol induced GES-1 cells (P<0.01). CONCLUSION: TA has a significant protective effect on alcohol damaged GES-1 cells, and its mechanism is closely related to activating the Keap-1/Nrf2 pathway, reducing oxidative stress, inhibiting the activation of the NLRP3 inflammasome pathway, alleviating inflammatory response, and thereby suppressing the activation of the mitochondrial apoptosis pathway.

Key words: tormentic acid, gastric cell damage, oxidative stress, inflammatory response, Keap1/Nrf2 signaling pathway, NLRP3 inflammasome signaling pathway

CLC Number:

R285.5

Rufeng ZHOU, Ying ZHANG, Ziyuan MENG, Jie LIU, Mengqiong SHI, Yong LIAO, Jie XU, Haoran LI, Yanqing TAN, Huilin QIN, Liangliang JIA, Yan YOU. Tormentic acid protects alcohol induced GES-1 cells via regulating Keap-1/Nrf2 and NLRP3 inflammasome signaling pathways[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(8): 1034-1052.

Figures/Tables 13

Fig.1 TA?s chemical structure and effects of different concentrations of TA and alcohol on the viabilities of GES-1 cells ($ \overline{x} $±s, n=5) A: TAʼs chemical structure; B: effect of TA on the viability of GES-1 cells; C: effect of ethanol on the cell viability of GES-1 cells; D: effect of ethanol on the cell viability of GES-1 cells induced by alcohol; bP<0.05, cP<0.01, compared with control group; eP<0.05, fP<0.01, compared with model group.

Table 1 Primer sequences

Gene	F:(5′-3′)	R:(5′-3′)
Keap-1	AGCGTGGAGAGAGATATGAGCC	ATCATCCGCCACTCATTCCT
Nrf2	GGCTACGTTTCAGTCACTT	AGCTCCTCCCAAACTTGCTC
GCLC	TGGCAATGCAGTGGTGGAT	AACACACCTTCCTTCCCATTGA
HO-1	TGGCAGCTGTCTCAAACCTC	TGCCGTTAAACACCTCCCTC
NQO1	CAGTGGTTTGGAGTCCCTGC	CAGTGGTTTGGAGTCCCTGCA
TXNIP	CAGCAGTGCAAACAGACTTCGG	CTGAGGAAGCTCAAAGCCGAAC
NEK7	ATGGAGGAGCCCGAGAAGAG	CTCCTGCTCCAGCTTCTTCC
NLRP3	GCACTGTGTTTGGAGTGGGTTTCA	CCCGACAGTGGATATAGAACAGA
ASC	TGGATGCTCTGTACGGGAAG	CCAGGCTGGTGTGAAACTGAA
Caspase-1	TTTCCGCAAGGTTCGATTTTCA	GGCATCTGCGCTCTACCATC
COX-1	CTATCACTGGATCCGTTCAT	AAAGTTCCTACCCCCACCAAT
COX-2	CAAATCCTTGCTGTTCCCACCCAT	GCACTGTGTTTGGAGTGGGTTTCA
iNOS	GCAATGGGCAGACTCTGAAG	CGATGTCATGAGCAAAGGCA
PGE2	CTCCTTGCCTTTCACGATTT	ACAACAGAGGACTGAACGCAT
Bcl-2	CATGTGTGTGGAGAGCGTCAA	GCCGGTTCAGGTACTCAGTCA
Bcl-xl	TCCTTGTCTACGCTTTCCACG	GGTCGCATTGTGGCCTTT
Bax	ATGGGCTGGACATTGGAC	GGGACATCAGTCGCTTCAG
Bad	CCAGAGTTTGAGCCGAGTGAG	GATGATGCTTGCCGGAGCC
Apaf-1	AGGACACAGACGGTGGAAAC	ATCGCACTGACCAGCTTCTT
GAPDH	AACGGCACAGTCAAGGCTGA	ACG CCAGTAGACTCCACGACAT

Fig.2 Effect of TA on migration ability of GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: cell scratch assay for detecting cell migration (scale bar=50 µm); B: transwell assay for detecting cell migration (scale bar=100 µm); cP<0.01, compared with control group; eP<0.05, fP<0.01, compared with model group.

Fig.3 Effect of TA on apoptosis of GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) cP<0.01, compared with control group; fP<0.01, compared with model group.

Fig.4 Effects of TA on ROS level and MMP of GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: ROS levels (scale bar=100 µm); B:MMP (scale bar=100 µm); cP<0.01, compared with the control group; fP<0.01, compared with the model group.

Fig.5 Effect of TA on cytokine levels of the supernatant of GES-1 cells and GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: cytokine levels in cell supernatant; B: mRNA expressions of cytokines in cells; cP<0.01, compared with control group; fP<0.01, compared with model group.

Fig.6 Effects of TA on LDH level and oxidative stress index of GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: LDH release; B: MDA levels; C-G: endogenous antioxidant enzyme activity; cP<0.01, compared with control group; fP<0.01, compared with model group.

Fig.7 Effect of TA on co localization of NLRP3 inflammasome in GES-1 cells induced by alcohol (scale bar=200 μm)

Fig.8 Effect of TA on the ultrastructure in GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: mitochondrial ultrastructure of GES-1 cells under electron microscopy; B: quantitative comparison of mitochondrial area; C: comparison of mitochondrial count, with each count defined within the size range of 4 μm × 4 μm; D: comparison of mitochondrial aspect ratio, i.e. the ratio of mitochondrial long axis to short axis; The red arrow points to the mitochondria, and the yellow arrow points to the nucleus, scale bar=2 μm; cP<0.01, compared with control group; fP<0.01, compared with model group.

Fig.9 Effects of TA on the relative gene mRNA and protein expressions Keap1/Nrf2 pathway in GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: relative gene mRNA expressions of Keap-1/Nrf2 pathway; B: relative gene protein expressions of Keap-1/Nrf2 pathway; cP<0.01, compared with control group; eP<0.05, fP<0.01, compared with model group.

Fig.10 Effects of TA on the relative gene mRNA and protein expressions NLRP3 inflammasome pathway in GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: relative gene mRNA expressions of NLRP3 inflammasome pathway; B: relative gene protein expressions of NLRP3 inflammasome pathway; cP<0.01, compared with the control group; fP<0.01, compared with the model group.

Fig.11 Effects of TA on the relative gene mRNA and protein expressions of mitochondria-dependent apoptotic pathway in GES-1 cells induced by alcohol ($ \overline{x} $±s, n=5) A: cytochrome C content in cytosol and mitochondria; B: relative gene mRNA expressions of mitochondria-dependent apoptotic pathway; C: relative gene protein expressions of mitochondria-dependent apoptotic pathway; cP<0.01, compared with control group; fP<0.01, compared with model group.

Fig.12 The mechanism of TA regulating Keap-1/Nrf2 and NLRP3 inflammasome signaling pathways in protecting against ethanol induced GES-1 cell damage

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