Liu et al. BMC Molecular and Cell Biology(2023) 24:28
https://doi.org/10.1186/s12860-023-00489-y

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Glycyrrhizin inhibits LPS-induced
inflammatory responses in goat ruminal
epithelial cells in vitro
Junfeng Liu1,2†, Bei Ma1†, Guang Hao3, DuoDuo Su1, Tianyang Wang1, Ze Ding1 and Xuefeng Guo1*
Abstract
Inflammation plays a crucial role in the progression of Subacute Ruminal Acidosis (SARA). The experiment was
designed to investigate anti-inflammatory effects of glycyrrhizin on goats ruminal epithelial cells (GREC) whichwere induced SARA by Lipopolysaccharide (LPS) in vitro. The GREC were induced SARA by adding LPS at theconcentration of 5 μm and glycyrrhizin was added at different concentration of 0, 60, 90, 120, 150 μm. The
structural integrity of LPS-induced GREC with the treatment of glycyrrhizin were observed by electron microscope;
The levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured by ELISA; The number of Zo-1
and Occludin were measured, the expression of tight junction protein Occludin were measured by Western blot,
and the mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured in vitro. The results showedthat higher concentration treatment of glycyrrhizin led to better morphology in LPS-induced GREC. Glycyrrhizin
inhibited the growth of inflammatory factors TNF-α, IL-1β, IL-6, IL-8 and IL-12 in a dose-dependent manner.
The number of ZO-1 and Occludin increased with the increase of adding of glycyrrhizin. Western blot analysis
showed that the expression of tight junction protein Occludin in LPS-induced GREC increased with the addingof glycyrrhizin in a dose-dependent manner. Furthermore, the mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8
and IL-12 decreased significantly with the increase treatment of glycyrrhizin. Glycyrrhizin significantly inhibits LPSinduced inflammatory mediators in GREC and the effects are better with the increase treatment of glycyrrhizin in
vitro.
Keywords SARS, GREC, Glycyrrhizin, Inflammatory mediators, mRNA expression†

Junfeng Liu and Bei Ma contributed equally to this work.

*Correspondence:
Xuefeng Guo
gxfdky@126.com
1
College of Animal Science and Technology, Tarim University, Alar 843300,
Xinjiang, PR China
2
Department of Clinical Veterinary Medicine, College of Veterinary
Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR
China
3
Ordos Supply and Marketing Cooperative Logistics Service Centre,
Ordos 017000, Inner Mongolia, PR China

Introduction
With the growing intensity of feeding and the increasing demand for animal products, SARA has become oneof the most harmful and common diseases in ruminants
breeding [1]. Researches have showed that the incidenceof SARA in Italy, Ireland, Netherlands and the UnitedStates was 33%, 13.8%, 11%, 19-20.1% respectively [2–5].
Yamamoto et al. [6]. Reported that when fattening cattle
suffered from SARA, the rumen would be in the low acidenvironment for a long time, causing ruminal mucosa
shedding. SARA has been reported to increase the death
rate of ruminal epithelial cells and impressively damage© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

barrier function of rumen epithelium [7]. The content of
free LPS in the rumen increases sharply under the condition of SARA. LPS is the main toxin factor of Gram
negative bacteria, when the host was infected by Gram
negative bacteria, the monocytes and macrophages in
the host could rapidly recognize LPS and thus stimulated the release of inflammatory factors such as TNF-α,
IL-1β and IL-6 [8]. Ruminal epithelial cells play an important role in defensing against bacteria, but when theruminants were under the condition of SARA, the barrier function of rumen epithelium was injured, thus LPS
and inflammatory mediators could enter the blood fromrumen epithelium causing systemic inflammatory reaction and infection [9]. The mechanism of LPS-induced
systemic inflammatory response and infection is as follows: The LPS leads to the activation of TLR4 signaling
pathway, causing the release of inflammatory cytokines.
Meanwhile, When NF-κB, which has certain biological
functions in cell survival, inflammation, congenital andadaptive immunity [10], was activated by LPS, it would
be activated and the inflammatory mediators such as
TNF-α, IL-1β would be released. A LPS-induced SARA
model was established in vitro by our experiment using
goats epithelial cells previously and the optimal inflammatory concentration of LPS was 5 μm. Glycyrrhizin,
isolated from licorice, has been reported to have antioxidative [11, 12], anti-inflammatory [13, 14], antiviralsuch as hepatoprotection and detoxification [15, 16], andimprove immunity [17] effects. In animal production,some kinds of diseases can be prevented and the meat
quality of livestock can be improved by using glycyrrhizin properly [18]. However, there was few researches
investigating on effects of glycyrrhizin on LPS-induced
GREC. In this experiment, LPS-induced GREC were
made, and different concentration of glycyrrhizin wasadded to investigate effects of glycyrrhizin on inflammatory mediators in vitro, the purpose of the experiment
was to reveal the mechanism of glycyrrhizin inhibit on
LPS-induced GREC and to provide scientific evidence forusing glycyrrhizin to prevent and treat ruminants under
SARA.

Materials and methods
Reagents

Glycyrrhizin (purity > 99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (China). LPS waspurchased from Sigma (St. Louis, MO, USA). ELISA kitsfor TNF-α, IL-1β, IL-6, IL-8 and IL-12 were purchasedfrom Nanjing Institute of Bioengineering (China). BCA
protein quantitative kit and SYBR Green PCR kit werepurchased from Smer Fell Science and Technology Co.,
Ltd. (Shanghai, China). Reverse transcription kit waspurchased from Ferments. RIPA, Acrylamide(29: 1), TrisHCl pH = 8.8 electrophoretic buffer, Tris-HCl pH = 6.8

Page 2 of 8

electrophoretic buffer, SDS, TEMED, PBS phosphate buffer and DEPC were purchased from JRDUN Biotechnology (Shanghai) Co., Ltd. Alexa Fluor 488 Labeled goatanti-rabbit IgG (H + L), DAPI, Goat anti-rabbit-HRP secondary sera, Donkey anti-goat-HRP secondary sera andGoat anti-mouse-HRP secondary sera were purchasedfrom Biyuntian co., Ltd. Occludin(rabbit)and ZO-1 (rabbit) were purchased from Bioss. RMPI-1640 was purchased from Hyclone.
Cell grouping and processing

GREC were acquired from two Boer goats (6 weeksage). Two Boer goats were provided by the Experimental Animal Center of Huazhong Agricultural University(Wuhan, China). Goats were breed in separate cages(24 ± 1 °C, 65% relative humidity) and were free to ingest
foods and water. LPS-induced GREC were formulated
by adding 5 μm LPS into GREC primary cell culture for
2 h, after inducing, the cells were divided into five groups
with the treatment of glycyrrhizin of 0, 60, 90, 120 and
150 μm for 24 h, then cells or the supernatant was collected for further analysis.
Detection of structural integrity of LPS-induced GREC

The fresh LPS-induced GREC with the treatment of glycyrrhizin were washed by PBS for one time, fixed by glutaraldehyde, osmic acid, each fixing followed washing by
PBS for three times, dehydrated with different gradientsof ethanol 30%, 50%, 70%, 90%, 95% and 100%, each timelasted for 10–15 min, replaced with propylene epoxide
for two times, each time last for 10 min, permeated by
the ratio of propylene oxide to epon resinand 2:1 and 1:2
for 2 h respectively, whole epon resinand for the night,
polymerizates at 37 ℃ for 12 h, 45 ℃ for 24 h and 60℃ for 48 h, and finally, the samples were made into cellslice and observed by Transmission Electron Microscope(JEM-2100 F JEOL).
Autophagy examination

First fixed with 2.5% glutaraldehyde for 2 h. Fix with 2%
osmium acid for 2 h. Dehydrate with 50%, 70%, 80% and
90% ethanol gradients for 15 min each, and then dehydrate with 100% ethanol 3 times for 20 min each. Replacewith acetone 2 times for 15 min each. Impregnate withacetone: embedding agent = 1:2 for 4 h; impregnate withpure embedding agent 2 times for 4 h each. Place thesamples in the embedding plate containing pure embedding agent. The plates were polymerized at 65℃ for morethan 48 h each. The embedding head was trimmed intoa trapezoidal shape and the surface area of the sample
was less than 0.2 mm × 0.2 mm. Slicer Sects. 50–70 nm.
3% uranyl acetate-lead citrate double staining. Observe
the formation of autophagic structures, mainly including

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

Table 1 The primer sequence of targeted genes
Gene
NF-κB(p65)(XM-018043384.1)
TNF-α(NM-001286442.1)
IL-1β(XM-013967700.2
IL-6(NM-001285640.1)
IL-8(XM-005681749.3)
IL-12(NM-001285700.1)
GAPDH(XM-005680968.3)

Primer sequences (5’-3’)
F: GTGTAAAGAAGCGGGACTTGG
R: GCGGTTGTCAAAGATGGGATG
F: CCACGTTGTAGCCAACATCAG
R: AGATGAGGTAAAGCCCGTCAG
F: CCACCTCCTCTCACAGGAAATG
R: GATACCCAAGGCCACAGGAATC
F: TACCTGGACTTCCTCCAGAAC
R: CGAATAGCTCTCAGGCTGAAC
F: TCTTGGCCGCTTTCCTGCTC
R: TTTCGCAGTGTGGCCCACTCF: GACCAAACCTCAGCCAAAG
R: GACACAGATGCCCATTCAC
F: GCCCTGAGGCTCTCTTCCA
R: GCGGATGTCGACGTCACA

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Table 2 The system of reverse transcription reaction
Length (bp)
203
134
100
245
162
104
101

the morphology of autophagosomes and autophagic
lysosomes.

Reagents
RNA-Primer Mix
5×RT Reaction Buffer
25 mM dNTPs
25 U/µL RNase Inhibitor
200 U/µL M-MLV Rtase
Oligo(dt)18
ddH2O (DNase-free)
Total volume

Addition
12 µL
5 µL
1 µL
1 µL
1 µL
1 µL
4 µL
25 µL

Table 3 The system of PCR reaction
ReagentsSYBRGreen Mix
Upstream primer F
Downstream primer R
ddH2O
cDNA templet
Total volume

Addition12.5 µL
0.5 µL0.5 µL9.5 µL
2 µL
25 µL

Inflammatory mediators assay

The LPS-induced GREC and the treatment of glycyrrhizin mentioned above were centrifuged at 3000 r/min for
20 min and the supernatant was collected. The level of
TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured using
ELISA kits according to the manufacture’s instruction(Biolegend, Camino Santa Fe, CA, USA).
The number of Occludin and ZO-1

Primers design and synthesis

According to the mRNA sequences of NF-κB, TNFα, IL-1β, IL-6, IL-8 and IL-12 of goats in GenBank, theupstream and downstream primers of mRNA were
designed by Primer 5.0 [19, 20]. Primers were synthesized by JRDUN Biotechnology (Shanghai) Co., Ltd., asshown in (Table 1).

The LPS-induced GREC (5 × 105 cells/well) were seeded
in 24 well plates. Glycyrrhizin of different concentration
was subsequently treated in culture medium for 24 h,cells were washed with PBS for three times, each time
last for three minutes. The wells were fixed by formaldehyde, permeabilized by Triton X-100, blocked by BSA for
1 h and incubated with primary antibody and secondary
antibody, each process with washing with PBS for threetimes, and finally, the wells were sealed and taken photos
by fluorescence microscope.

RNA extraction and reverse transcription

Western blot analysis

Then the cDNA was subjected to PCR amplification after
autoclaving and diluting to 10 μm with distilled waterwith SYBR Green PCR kits (Thermo, #K0223). The reaction system was shown in (Table 3) and response procedure was that: 95 ℃, 10 min; (95 ℃, 15 s; 60 ℃, 45 s) ×40
cycles; 95 ℃, 15 s; 60 ℃, 1 min; 95 ℃, 15 s; 60 ℃, 15 s.

The LPS-induced GREC with the treatment of adding
glycyrrhizin were disrupted sufficiently at 4 °C, centrifuged at 12 000 g for 10 min and the supernatant wascollected. Total proteins were extracted with the protein
extraction reagent and the concentration was measured
with the BCA protein reagent. The protein was separated by SDS/PAGE and electrophoretic ally transferred
onto a semi-dry rotating membrane. The membrane wasblocked in NaCl/Tris containing 5% nonfat dry milk atroom temperature for 1 h, and incubated with a primary
antibody at 4 ℃ for 12 h and then incubated with a secondary antibody at 37 ℃ for 1 h. Finally, the blots weredeveloped with the ECL Plus Western Blotting Detection
System (GE Healthcare, Chalfont St Giles, UK).

The total RNA was extracted by using Trizol (Invitek,
1596 − 206). The first strand of cDNA was synthesizedaccording to the instructions of Dalian Bao Biologicalengineering Co., Ltd and was synthesized according to
the reverse transcription kit (Fermentas, #K1622) [21].
The reaction system was showed in (Table 2). The reverse
transcription was carried out at 37 ℃ for 60 min, 85 ℃
for 5 min and 4 ℃ for 5 min.
Real-time PCR

The mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and
IL-12

After Real-time PCR amplification, the relative mRNA
expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12
were measured with standard mRNA curve.

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

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Fig. 1 (A-E). Effects of glycyrrhizin on structural integrity in LPS-induced GREC. Observation of morphological changes in different stages of autophagy
using transmission electron microscopy. Figure 1 A, B, C, D and E was investigated at the concentration of glycyrrhizin was 0, 60, 90, 120, 150 μm respectively. The autophagy around the nucleus was the highest in picture A, and gradually decreased in picture B, C, D and E

Fig. 2 (A-E). Effects of glycyrrhizin on inflammatory mediators TNF-α (A), IL-1β(B), IL-6(C), IL-8(D) and IL-12(E) in LPS-induced GREC. The data presented
were the means ± S.E.M of three independent experiments. **p < 0.01, *p < 0.05 versus LPE-induced GREC without treatment of glycyrrhizin

Statistical analysis

All results were expressed as the means ± SEM with SPSS
16.0. Comparisons between groups were performed withANOVA followed by Duncan test. Differences were considered to be significant at P < 0.05 or *, extremely significant at P < 0.01 or **.

Results
Effect of glycyrrhizin on structural integrity of LPS-induced
GREC

Qualitative examination, LPS-induced GREC in the cytoplasm, autophagic vesicles and autophagic lysosomes
were formed; including crescentic or cup-shaped, bilayer
or multilayer membranes; bilayer or multilayer membranes with vesicle-like structures, etc. Monolayers withdegraded cytoplasmic components. As shown (Fig. 1),
the autophagy is most evident in Fig. 1A. In Fig. 1B, C, D

and E, autophagy decreases in a dose-dependent mannerwith the addition of glycyrrhizin; and the best cell morphology of GREC is observed when the glycyrrhizin concentration is 150 μm.
Effects of glycyrrhizin on inflammatory mediators in LPSinduced GREC

The effects of glycyrrhizin on LPS-induced TNF-α, IL-1β,
IL-6, IL-8 and IL-12 were measured to investigate theanti-inflammatory effects of glycyrrhizin. As showedin (Fig. 2A, B, C, D, E), the production of inflammatory
mediators in LPS-induced GREC without treatment ofglycyrrhizin was significantly higher than those with thetreatment of glycyrrhizin (P < 0.01) and the treatment ofglycyrrhizin inhibited LPS-induced inflammatory mediators production in a dose-dependent manner.

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

Effects of glycyrrhizin on the number of protein of ZO-1
and Occludin in LPS-induced GREC

To further confirm effects of glycyrrhizin on LPSinduced GREC, the number of Occludin and ZO-1 wasmeasured and showed in (Fig. 3A, B). With the increaseof glycyrrhizin, the number of Occludin and ZO-1 significantly increased and the number of Occludin and ZO-1
was the most when the concentration of glycyrrhizin was
150 μm.
Effects of glycyrrhizin on the expression of Occludin

The content of Occludin was shown in Fig. 4, with theincrease treatment of glycyrrhizin, the content of Occludin gradually increased and the concentration reachedthe highest when the concentration of glycyrrhizin was
150 μm.
Effects of glycyrrhizin on mRNA expression of NF-κB, TNFα, IL-1β, IL-6, IL-8 and IL-12 in LPS-induced GRECAs showed in Fig. 5A, B, C, D, E, F; the mRNA expression
of NF-κB, TNF-α, IL-1 β, IL-6, IL-8 and IL-12 in LPSinduced GREC was extremely higher than those withthe treatment of glycyrrhizin (P < 0.01) and glycyrrhizin
inhibited the mRNA expression of these inflammatory
cytokines in a dose-dependent manner. Effects of inhibition were the best when the concentration of glycyrrhizin
was 150 μm.

Discussion
There are many researches investigating on the antiinflammatory and antiviral effects of Glycyrrhiza glabra[22], which has been used to treat disorders like endometriosis and mastitis. Glycyrrhizin is the major ingredientof Glycyrrhiza glabra. Wang et al. [23] reported that glycyrrhizin could inhibit LPS-induced inflammatory mediators in endometrial epithelial cells. Inflammation plays acrucial role in the progression of SARA. It was reported
that when the ruminants were under SARA, the LPS

Page 5 of 8

and inflammatory mediators increased. Emmanuel et al.
[24]. Reported that when the level of LPS was 5 µg/mL
in rumen in vitro, the permeability of rumen increased,
LPS easily penetrated the rumen wall into the blood. In
this experiment, effects of glycyrrhizin on LPS-induced
GREC were investigated. The integrity of cells are very
important for cells to be live. In this experiment, thestructural integrity of LPS-induced GREC became better with the treatment of glycyrrhizin and the structural
integrity of LPS-induced GREC become well with theincrease adding of glycyrrhizin, indicating that glycyrrhizin could protect and treat the cellular morphology of
LPS-induced GREC.
When the body is infected by external bacteria, inflammatory cytokines that regulate and control inflammatory
responses are released, including TNF-α, IL-1β, IL-6,
IL-8 and IL-12. LPS induced monocyte phagocytes torelease IL-1, IL-6, TNF-α and other inflammatory factors after entering the blood to participate in circulation.
In this experiment, the results showed that glycyrrhizin
successfully inhibited inflammatory mediators in a dosedependent manner, which was consistent with Fu et al.
[25].
The rumen epithelium from mucous layer to serosa
was stratum carenum, stratum granulosum, and stratum
spinosum and stratum basales. There is tight connection
protein in stratum granulosum, which plays an important role in maintaining rumen barrier function [26, 27].
The tight junction protein are composed of Claudin protein, Occluding protein, ZO-1, ZOs and so on. Sun et al.
[28]. reported that when the epithelial cells were under
SARA, pH interacted with LPS or HIS worked on rumen
epithelium and decreased the mRNA expression of tightjunction protein in rumen epithelium in vitro. When theruminal epithelial cells were induced by LPS, the tightjunction protein was destroyed, the cell gap became
larger. By investigating, the number of the tight junction
proteins can determine whether pathological changes

Fig. 3 (A-B). Effects of glycyrrhizin on the number of protein of ZO-1(A) and Occludin (B). The number of the ZO-1(the blue color was nucleus and thegreen is ZO-1) and Occludin (the blue color was nucleus and the green is Occludin, ×400, scale = 50 μm)). From left to right the LPS-induced GREC were
investigated at the adding concentration of glycyrrhizin at 0, 60, 90, 120, 150 μm. The number of ZO-1 and Occludin increased with the increase addingof glycyrrhizin

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

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Fig. 4 Effects of glycyrrhizin on the expression of Occludin. Apply Image Pro Plus (IPP) 6.0 software to calculate the grayscale value of the protein. β-actin
was used as control. Blots were cut prior to hybridization with the antibody. Include images of all blots and all duplicate images in the supplement

Fig. 5 (A-F). Effects of glycyrrhizin on the mRNA expression of NF-κB (A), TNF-α (B), IL-1β(C), IL-6(D), IL-8(E) and IL-12(F). Glycyrrhizin decreased the mRNA
expression of inflammatory mediators in a dose-dependent manner. The data presented were the means ± S.E.M of three independent experiments. **p < 0.01, * p < 0.05 versus LPS-induced GREC without treatment of glycyrrhizin

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

had occurred in rumen epithelium. In this experiment,
immunofluorescence was used to investigate the distribution of Occludin and ZO-1. The number of Occludin and ZO-1 increased significantly in LPS-induced
GREC with the treatment of glycyrrhizin. The results
further verified that glycyrrhizin protected rumen epithelium and restored the function of cell barrier. TNF-α
is an anti-tumor multi-function factor which can promote the tumor necrosis and a large amount of TNF-α
can cause serious inflammatory reactions such as bloodpressure increase and tissue necrosis. TNF-α is the main
signal transduction molecule of LPS in organism reaction and it can inhibit the expression of Occludin largely[29–31].Thus, the content of Occludin was measured inthis experiment to investigate on whether glycyrrhizin
could increase the content of Occludin. Results showedthat the content of Occludin in LPS-induced GREC wasa little, and the content of Occludin increased with thetreatment of glycyrrhizin in a dose-dependent manner.
These results may indicate that glycyrrhizin could inhibit
the expression of TNF-α, which needs to be further
investigated.
Stimulating ruminal epithelial cells with LPS lead tothe activation of NF-κB signaling pathway [32]. NF-κB
signaling pathway has attracted widespread attention
for its ability to regulate transcription of inflammatory
mediators [33]. By activating NF-κB, LPS can increase
the transcription of TNF-α, IL-1β and IL-6, inducing theexpression of pro-inflammatory response mediators [34].
NF- κB is considered to be an important pathogenic factor in many acute and chronic inflammatory diseases,
thus increase the efficiency of inhibiting NF-κB can effectively treat inflammatory diseases [35]. Effects of glycyrrhizin on mRNA expression of TNF-α, IL-1β, IL-6, IL-8
and IL-12 in NF-κB signaling pathway in LPS-induced
GREC were investigated and the results showed thatwith the increase treatment of glycyrrhizin, the mRNA
expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12
decreased significantly. The results showed that glycyrrhizin inhibited the LPS-induced inflammatory mediators at the genetic level.
However, in this experiment, glycyrrhizin inhibited LPS
induced cellular inflammatory damage; And its impact
on cell autophagy. In the future, animal experiments will
reveal the ability of glycyrrhizin to inhibit inflammation
and autophagy.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00489-y.
Supplementary Material 1

Page 7 of 8

Acknowledgements
The authors thank all staff, graduate students at the Center for Reproductive
Medicine for their support in the preparation of this manuscript. A preprint
has previously been published [35].
Author contributions
Junfeng Liu and Xuefeng Guo contributed to the conception of the study;
Ze Ding and Tianyang Wang performed the experiment; Junfeng Liu andDuoDuo Su contributed to wrote the manuscript; Guang Hao contributed
to the analysis of the experimental data; Bei Ma contributed to the Writingoriginal draft and Writing–review editing; Junfeng Liu and Xuefeng Guo
helped perform the analysis with constructive discussions. The author(s) readand approved the final manuscript.
Funding
Fund of the President of Tarim University (No. TDZKCX202209); National
Natural Science Foundation of China (No. 32260905). University student
innovation and entrepreneurship training program (No.202210757001).
Data Availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
All the experiments were performed in accordance with relevant guidelines
and regulations. The experiments performed on animals were according to
the ARRIVE guidelines. All experimental protocols were approved by Ethics
Committee of Huazhong Agricultural University (HZAUMO-2015‐12), Wuhan,
China.
Consent for publication
Not Applicable.
Received: 3 February 2023 / Accepted: 4 September 2023

References
1. Plaizier JC, Mulligan FJ, Neville EW, et al. Invited review: Effect of subacuteruminal acidosis on gut health of dairy cows. J Dairy Sci. 2022;105(9):7141–60.
2. Morgante M, Stelletta C, Berzaghi P, et al. Subacute rumen acidosis in lactating cows: an investigation in intensive italian dairy herds. J Anim Physiol
Anim Nutr. 2007;91(5–6):226–34.
3. Kleen JL, Hooijer GA, Rehage J, et al. Subacute ruminal acidosis in dutch dairy
herds. Vet Rec. 2009;164(22):681–3.
4. O’Grady L, Michael L, Finbar J. Subacute ruminal acidosis (SARA) in grazing
irish dairy cows. Vet J. 2008;176(1):44–9.
5. Ma J, Shah AM, Wang Z et al. Potential protective effects of thiamine supplementation on the ruminal epithelium damage during subacute ruminal
acidosis. Anim Sci J, 2021, 92(1).
6. Wijnands AM, de Jong ME, Lutgens MWMD, et al. Prognostic factors foradvanced colorectal neoplasia in inflammatory bowel disease: systematic
review and meta-analysis. Gastroenterology. 2021;160(5):1584–98.
7. Fiore E, Faillace V, Morgante M et al. A retrospective study on transabdominal
ultrasound measurements of the rumen wall thickness to evaluate chronic
rumen acidosis in beef cattle. BMC Vet Res, 2020, 16(1).
8. Yan Z, Effect. Mechanism and function analysis of LPS treatment on FTO
expression in broiler chickens. Agricultural Univ Nanjing 2014.
9. Fu Y, He Y, Xiang K, et al. The role of rumen microbiota and its metabolites insubacute ruminal acidosis (SARA)-induced inflammatory diseases of ruminants. Microorganisms. 2022;10(8):1495.
10. Rietschel ET, Brade H, Brade L et al. Lipid A, the endotoxic center of bacterial
lipopolysaccharides: relation of chemical structure to biological activity. Prog
Clin Biol Res. 1987;25–53.

Liu et al. BMC Molecular and Cell Biology(2023) 24:28

11. Haraguchi H, Ishikawa H, Mizutaki K, et al. Antioxidative and superoxide scavenging activities of retrochalcones in Glycyrrhiza inflata Bioorg. Med Chem.
1998;6(3):339–47.
12. Yang Y, Liu Y, Feng Z, et al. Seven new flavonoid glycosides from the rootsof Glycyrrhiza uralensis and their biological activities. Carbohydr Res.
2019;485:107820.
13. Yamashita T, Asano Y, Taniguchi T, et al. Glycyrrhizin ameliorates fibrosis, vasculopathy, and inflammation in animal models of systemic sclerosis. Investig
Dermatol. 2017;137(3):631–40.
14. Gonzalez-Reyes S, Santillan-Cigales J, Jimenez-Osorio A, et al. Glycyrrhizinameliorates oxidative stress and inflammation in hippocampus and olfactory
bulb in lithium/pilocarpine induced status epilepticus in rats. Epilepsy Res.
2016;126:126–33.
15. Okamoto T, Kajino K, Hino O. Hepatoprotective drugs for the treatment
ofvirus-induced chronic hepatitis: from hypercarcinogenic state to hypocarcinogenic state. Jpn J pHarmacol. 2001;87(3):177–80.
16. Shibayama Y. Prevention of hepatotoxic responses to chemicals by glycyrrhizin in rats. Exp Mol Pathol. 1989;51(1):48–55.
17. Jue L, Xue G. Effect of glycyrrhizin on immunoglobulin ig A and ig G in bloodof karakur sheep. Jiangsu Agricultural Science. 2014;42(1):172–4.
18. Lee SA, Lee SH, Kim JY, et al. Effects of glycyrrhizin on lipopolysaccharide-induced acute lung injury in a mouse model. J Thorac Disease.
2019;11(4):1287–302.
19. Ting W. Effect of rumen histamine on inflammatory pathway of rumen
epithelial cells in cattle with subacute rumen acidosis. Jilin Univ. 2015.
20. Hui W, Hua Z, Ying S. Effect of lipopolysaccharide on the expression of TLR2
and TLR4 in intestinal mucosa microvascular endothelial cells. China Agricultural Bulletin. 2015;31(11):72–7.
21. Gui Z, Xiao C, Rong C, et al. Expression of NF-κB LPS-induced in bovine endometrial epithelial cell. Chin Veterinary Sci. 2010;40(10):1063–6.
22. Uto T, Morinaga O, Tanaka H, Shoyama Y. Analysis of the synergistic effect ofglycyrrhizin and other constituents in licorice extract on lipopolysaccharide
induced nitric oxide production using knock-out extract. Biochem Biophys
Res Commun. 2012;417(1):437–78.
23. Xue W, Hong H, Lan C. Glycyrrhizin inhibits LPS-induced inflammatory
mediator production in endometrial epithelial cells. Microb Pathog.
2017;109:110–3.

Page 8 of 8

24. Emmanuel D, Dunn S, Ametaj B. Feeding high proportions of barley
grain stimulates an inflammatory response in dairy cows. J Dairy Sci.
2008;91(2):606–14.
25. Fu YH, Zhou ES, Wei ZK, et al. Glycyrrhizin inhibits the inflammatory response
in mouse mammary epithelial cells and a mouse mastitis model. FEBS J.
2014;281(11):2543–57.
26. Harhaj S, Antonetti A. Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biology. 2004;36(7):1206.
27. Steele M, Croom J, Kahler M, et al. Bovine rumen epithelium undergoes rapid
structural adaptation during grain-induced subacute rum inal acidosis. Am J
Physiology: Regul Integrativeand Comp Physiol. 2011;300(6):R1515–1523.
28. Yan S, Gao M, XU M, et al. Interaction effects of pH and lipopolysaccharide
or histamine on mRNA expression levels of tight junction proteins of rumen
epithelium of dairy goats in vitro. J Anim Nutr. 2018;30(5):1816–26.
29. Hong S, Sa L, Li M et al. Effect of TNF-α on tight junctions between theepithelial cells of intestinal mucosal barrier. Chin Dig J World. 2004;(6):49–52.
30. Bruewer M, Luegering A, Kucharzik T, et al. Proinflammatory cytokines disrupt
epithelial barrier function by apoptosis-independent mechanisms. Immunol.
2003;171(13):6164–72.
31. La X, Dan S, Yu Z, et al. Relationship between occludin expression in intestinal
epithelial cells and tumor necrosis factor-αlevel in rats with nonalcoholic fatty
liver disease. Chin Dig J World. 2010;18:981–6.
32. Schaefer T, Desouza K, Fahey J, et al. Toll-like receptor (TLR) expression and
TLR-mediated cytokine/chemokine production by human uterine epithelial
cells. Immunology. 2004;112(3):428–36.
33. Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors andits regulation. Acad Psychol Bull. 2009;1(4):a000034.
34. Bruewer M, Luegering A, Kucharzik T, et al. Proinflammatory cytokines disrupt
epithelial barrier function by apoptosis-independent mechanisms. J Immunol. 2003;171(11):6164–72.
35. Wullaert A, Bonnet MC, Pasparakis M. NF-κB in the regulation of epithelial
homeostasis and inflammation. Cell Res. 2011;21(1):146–58.

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