Gastric protective effect of Alpinia officinarum flavonoids: mediating TLR4/NF-κB and TRPV1 signalling pathways and gastric mucosal healing

Figures & data

Table 1. The corresponding antibody name, manufacturer and dilution multiple list in Western blot experiment.

Antibody name	Manufacturer	Dilution multiple
Anti-endothelin 1 antibody	Abcam (ab2786)	1:1000
Anti-iNOS antibody	Beyotime(AF7281)	1:1000
Anti-Bax antibody	Beyotime (AF1270)	1:2000
Anti-Bcl-2 antibody	Abcam (ab32124)	1:1000
Anti-caspase-3 antibody	Beyotime (AF1213)	1:2000
Anti-EGF antibody	Abcam (ab218831)	1:2500
Anti-TRPV1 antibody	SANTA (sc-398417)	1:500
Anti-TLR4 antibody	Abcam (ab217274)	1:1000
Anti-NF-κBp65 antibody	Abcam (ab16502)	1:1000
Anti-phospho-IKB alpha	CST (2859T)	1:1000
Anti-beta actin antibody	Abcam (ab8227)	1:1000
Goat anti-rabbit IgG H&L (HRP)	Abcam (ab97051)	1:20,000

Table 2. The mRNA primer sequence list for real-time quantitative PCR (qRT-PCR) in the experiment.

Primers	Forward/reverse	Sequence
Bcl-2	Forward	5′-CTGGTGGACAACATCGCTCT-3′
	Reverse	5′-ATAGTTCCACAAAGGCATCCCAG-3′
Bax	Forward	5′-GGTTTCATCCAGGATCGAGCA-3′
	Reverse	5′-TCCACATCAGCAATCATCCTCTG-3′
Caspase-3	Forward	5′-TTGGAACGGTACGCGAAGAA-3′
	Reverse	5′-AGAGTCCATCGACTTGCTTCC-3′
OSM	Forward	5′-TTAGTTTGGCCCTTGCGCT-3′
	Reverse	5′-GTTTTGGTGGAGGATATAGGGCT-3′
PDGFRB	Forward	5′-ACTATGTCCCATCTGCCCCT-3′
	Reverse	5′-ACTTGCCCTCACAGATGAGC-3′
CSF3R	Forward	5′-AGCCCCAATCATTGCTGAGA-3′
	Reverse	5′-AGCTTGGTGTTTACCTGCCT-3′
EGF	Forward	5′-TGCCAACTGGGGGTGCACAG-3′
	Reverse	5′-CTGCCCGTGGCCAGCGTGGC-3′
FGF18	Forward	5′-GCATCCATGTGGAGAACCAGA-3′
	Reverse	5′-CCACTAGGAGCTGGGCATACT-3′
SYK	Forward	5′-CCTGATGTGGGAAGCGTTCT-3′
	Reverse	5′-GGCCTGTTCTCCACATCGTA-3′
PGF	Forward	5′-TGAGGAACCCCACCTGTGAT-3′
	Reverse	5′-CTTGTCCTTCCATGCCCCTT-3′
NGF	Forward	5′-GCATCGCTCTCCTTCACAGAG-3′
	Reverse	5′-ACATTACGCTATGCACCTCAGAGT-3′
SRC	Forward	5′-TGAGGCATGAGAAGCTGGTG-3′
	Reverse	5′-TGCCTGAAGCAATCTGAGCA-3′
P2RY2	Forward	5′-CTGTCGACTCAGCGCCAAA-3′
	Reverse	5′-CTGGAGGGCTCCGAGGT-3′
TRPV1	Forward	5′-AGGGAGATCCATGAACCCGA-3′
	Reverse	5′-AGCATGTCATGACGGTTAGGG-3′
HTR2A	Forward	5′-GGTCATCATGGCAGTGTCCC-3′
	Reverse	5′-AAGGCCACCGGTACCCATAC-3′
BDKRB2	Forward	5′-ATACCGTGACCAGGACTTGAC-3′
	Reverse	5′-GTTGAACATTTCAATGCAGGTG-3′
CALCA	Forward	5′-CCTTTGAGGTCAACCTTGG-3′
	Reverse	5′-ATAGTTCTGCACCAGTGCA-3′
LBP	Forward	5′-ATGGAGATTGAAGGCTTTGTG-3′
	Reverse	5′-GAATACATTCGTGACCACGC-3′
CD14	Forward	5′-CGACCATGAAGCTTATGCTC-3′
	Reverse	5′-GAGAAGTTGCAGTAGCAGC-3′

Figure 1. Composition and structure of F.AOH. (A, B) (Lin et al. 2020) macroscopic appearance and histological evaluation of gastric mucosa in rats with acetic acid-induced gastric mucosal damage (D, E), (a, g) normal group; (c, i) positive control group ranitidine (100 mg/kg); (d, j) F.AOH low- (13.5 mg/kg); (e, k) medium- (27 mg/kg); (f, l) high-dose (54 mg/kg); (b, h) model group treated with acetic acid; flowchart of experiments (C); F.AOH can increase the ulcer inhibition rate and decrease ulcer index in rats with GU (F, G). ^##p < 0.01 compared with model group (n = 6).

Figure 2. The levels and expressions of endothelin-related inflammatory factors (A–C), the expression levels of nitric oxide synthase, and ET-1 proteins were analysed; (D–F) the levels of inflammatory factors NO, IL-6 and TNF-α in serum. **p < 0.01 compared with normal group; ^#p < 0.05 and ^##p < 0.01 compared with model group (n = 6).

Table 3. F.AOH attenuates oxidative stress in rats with gastric injury induced by acetic acid.

Group	MPO (U/mL)	SOD (U/mL)	GSH (nmol/mL)	MDA (nmol/mL)
Normal	1.52 ± 0.67	17.62 ± 0.98	41.6 ± 4.8	15.74 ± 1.37
RAN (100 mg/kg)	13.45 ± 1.02#	10.4 ± 0.7##	26.43 ± 6.45#	49.61 ± 3.63##
F.AOH (31.7 mg/kg)	11.28 ± 1.06#	6.9 ± 1.26	28.1 ± 5.2#	61.42 ± 6.2#
F.AOH (63.4 mg/kg)	8.47 ± 3.47##	8.31 ± 1.7#	30.3 ± 3.4##	41.7 ± 7.83##
F.AOH (126.8 mg/kg)	6.15 ± 0.84##	13.28 ± 1.54##	33.41 ± 4.38##	33.76 ± 3.84##
Acetic acid model	17.72 ± 1.42**	5.6 ± 1.2**	18.8 ± 3.46**	98.65 ± 5.52**

**p < 0.01 compared with normal group; ^#p < 0.05 and ^##p < 0.01 compared with model group (n = 6).

Figure 3. F.AOH promoted proliferation, migration and inhibiting apoptosis of GES-1 cells in vitro. (A, B) Flow cytometry and apoptosis rates for each group: (a) normal group, (b) positive control group ranitidine, (c) F.AOH low- (13.5 mg/kg), (d) medium- (27 mg/kg), (e) high-dose- (54 mg/kg), (f) model group treated with ethanol; (C) F.AOH can promote the penetration rate of GES-1 cells in transwell test: (a) normal group, (b) model group treated with ethanol, (c) positive control group ranitidine, (d) F.AOH low- (13.5 mg/kg), (e) medium- (27 mg/kg), (f) high-dose- (54 mg/kg); (D–H) F.AOH inhibiting the expression of Bcl-2/Bax/caspase-3 pathway proteins and mRNA in GES-1 apoptosis in vitro. *p < 0.05 and **p < 0.01 compared with normal group; ^#p < 0.05 and ^##p < 0.01 compared with model group.

Figure 4. (A) Venn maps of differential gene expression. The volcanic map of (B) NOR vs. AC and (C) AC vs. MD (red dots are upregulated genes, blue dots are upregulated genes and grey dots are non-significant differential genes).

Figure 5. The map of GO enrichment and KEGG pathway enrichment analysis of differential genes. (A, B) GO functional enrichment analysis map of NOR vs. AC and AC vs. MD (single gene bubble map of the top 20 enrichment abundance); (C, D) the KEGG functional enrichment analysis map of NOR vs. AC and AC vs. MD (single gene set bubble map of the top 30 enrichment abundance).

Figure 6. The 30 genes with the most significant differences among all differential genes in the first 30 signalling pathways enriched by KEGG of AC vs. MD.

Figure 7. PPI network recognition of key genes and Western blot and qPCR verification experiments. (A–L) PI3K-Akt pathway, Toll-like receptor pathway and inflammatory mediators regulate the relative expression levels of TRP channel-related genes and proteins. **p < 0.01 compared with normal group; ^##p < 0.01 compared with model group: values are expressed as means ± standard deviation (n = 3).

Figure 8. F.AOH is involved in TLR4/NF-κB and TRPV1 signalling pathways and gastric mucosal ulcer healing.

Lin KW, Wang Y, Gong JW, Tan YF, Deng T, Wei N. 2020. Protective effects of total flavonoids from Alpinia officinarum rhizoma against ethanol-induced gastric ulcer in vivo and in vitro. Pharm Biol. 58(1):854–862.

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