Introduction
Alcoholic
liver disease (ALD) is caused by long-term
heavy drinking (Li et al. 2011).
It usually manifests as fatty liver in the initial stage and develops into
alcoholic hepatitis, cirrhosis and hepatic fibrosis. There is a lack of
large-scale epidemiological survey data on alcoholic liver disease in China
(Wei et al. 2015; Ciardullo et al. 2020).
The
first-line therapy for severe alcoholic hepatitis is corticosteroids; however, the evidence for its effectiveness in reducing
mortality remains unclear.
Pentoxifylline is an alternative therapy (Mitchell). Succinyl rotundic acid has pharmacological effects
including antioxidation, anti-inflammation, scavenging oxygen free radicals,
and others. It treats liver diseases such as acute liver injury, nonalcoholic
fatty liver, liver fibrosis, liver cancer, and others. In view of these
problems, our research group designed succinyl rotundic acid (Li et al. 2017)
to treat alcoholic liver disease.
This study
established a chronic ethanol-induced liver injury model in rats, studied its
hepatoprotective effects, explored the relevant mechanisms, and measured liver
function and liver tissue oxidation injury indexes to evaluate the therapeutic
effects of succinyl rotundic acid (Hsu et al. 2015) on alcoholic liver
disease.
Materials
and Methods
Materials
sample
Succinyl
rotundic acid (SRA) was obtained by chemical modification of RA. RA was heated
in the presence of pyridine and reacted with succinic anhydride. Cooling to
room temperature, after the completion of an action in ice water, dilute
hydrochloric acid was added to adjust the pH to 4–. After filtration, the
filter residue was washed with water until it was colorless. The final SRA
product was obtained by recrystallizing. The purity of SRA was 95.70% by HPLC.
Sprague Dawley (SD) rats, male, weights 180–220 g were used. After
adaptive feeding, 60 SD rats were divided into normal, model, succinyl rotundic
acid low-dose (10 μmol/L),
middle-dose (20 μmol/L), and
high-dose (40 μmol/L) groups
according to body weight. In the normal group, the rats were given equal volume
distilled water. Except for the normal group, rats in each group were given 10
mL/kg of 60% ethanol solution orally every day. After 6 h, the drug group was
given the corresponding drug orally according to the designed dose (2.0 mL/kg-d)
for 30 days. The model group was not given drugs. After the final
administration, the rats were fasted for 16 h without withholding drinking
water, weighed, and blood and liver tissues were taken for each index test.
LDH,
AST, ALT,
NOS and NO measurement
Serum was
separated and levels of lactate dehydrogenase (LDH), aspartate transaminase
(AST) and alanine aminotransferase (ALT) were determined using an automatic
blood biochemical analyzer. The activities of nitric oxide synthase (NOS) and
nitric oxide (NO) were determined by a chemical chromogenic method according to
the kit instructions.
MDA,
GSH and TG
measurement
Liver tissue
was homogenized according to kit instructions. The levels of malondialdehyde
(MDA), glutathione (GSH) and triglyceride (TG) in liver tissue were measured
according to the kit instructions.
Western
blotting
Liver tissue
homogenates was prepared using RIPA to obtain protein samples. A BCA protein
concentration determination kit was used to measure protein concentrations.
After SDS-PAGE electrophoresis, proteins were transferred to polyvinylidene
fluoride (PVDF) membranes and washed in TBST. Membranes were incubated with
antibodies to Bcl-2 (1:700), Bax (1:2000), caspase-3 (1:700), NF-kB p 65
(1:2000), IKBA (1:2000), HO-1 (1:2 000), Nrf2 (1:2000), CYP2E1 (1:2000), and β-actin
(1:2000) overnight at 2–8ºC; then added goat anti-rabbit secondary antibody
(1:2000). Detection, exposure, development and fixation involved
ultra-sensitive enhanced chemiluminescence (ECL) reagents. Gel-Pro-Analyzer
software (Media Cybernetics Inc.) was used to measure the optical density.
Statistical
analysis
The data was
expressed as M ± S. Use Bonferroni post–hoc test for multiple comparisons and
one-way ANOVA was used for inter-group comparison. Use GraphPad Prism 5.0
software to process data and numbers.
Results
Body weight
The initial
body mass in each group of rats showed no significant differences before
intragastric administration. After 30 days of continuous gavage, the body
weights in the normal group were higher than those of other groups, however
there without significance (Table 1 and Fig. 1A).
Content
of LDH, AST, ALT, NOS and NO in liver
tissue
LDH, AST, NOS
and NO levels in the model group were significantly greater differences (P < 0.01, P < 0.05, P < 0.001,
and P < 0.001, respectively) than
those of the the normal group. Serum LDH levels in the low-, middle-, and
high-dose groups were significantly lower than those of the model group (P < 0.01, P < 0.001, and P <
0.001, respectively, Fig. 1B); AST content in serum was significantly lower in
the three groups (P < 0.05, P < 0.01, and P < 0.01, respectively, Fig. 1C). Serum ALT levels in the low-,
middle-, and high-dose groups were significantly lower (P < 0.05, P < 0.05
and P < 0.01, respectively, Fig.
2A). Serum levels of NOS and NO were significantly lower in the middle- and
high-dose groups (P < 0.05 and P < 0.01, respectively, Fig. 2B–C and
Table 2).
Changes
of MDA, GSH and TG in liver tissue
Levels of MDA
and TG in the model group were significantly higher, while levels of GSH were
significantly lower (P < 0.001, P < 0.05, and P < 0.01, respectively) than those of the normal group. MDA
levels in liver tissue were significantly lower in the middle-, and high-dose
groups (P < 0.05 and P < 0.01, respectively Fig. 3A), GSH
levels in liver tissue were significantly higher in the high-dose group (P < 0.05, Fig. 3B), TG levels in
liver tissue were significantly lower in the high-dose group (P < 0.05, Fig. 3C and Table 3) than
in the model group.
Western
blotting
Liver levels
of Bcl-2 in the model group were significantly lower (P < 0.001), and levels of Bax and caspase-3 were significantly
greater (P < 0.001 in all) than in
the normal group. Levels of Bcl-2 in liver in the high-dose group were
significantly greater (P < 0.01),
and levels of Bax and caspase-3 in the high- and middle-dose groups were
significantly lower (P < 0.01 and P < 0.05, respectively) (Fig. 4A–B)
than in the model group.
Western
blotting revealed that levels of CYP2E1 in liver tissue in the model group were
significantly greater than those of the normal group (P < 0.001). The high-, middle-, and low-dose groups showed
significantly lower levels of CYP2E1 (P
< 0.001, P < 0.01, and P < 0.05 respectively) (Fig. 4C–D)
than those of the model group. These findings suggest that succinyl rotundic
acid inhibits hepatic levels of CYP2E1 in rats with alcoholic hepatitis,
thereby promoting the rapid decomposition of ethanol and reducing
ethanol-induced damage to the liver.
Hepatic
levels of Nrf2 in the model group were significantly lower than those of the
normal group (P < 0.001). The
high-dose group showed significantly higher Nrf2 levels (P < 0.01) (Fig 4C–D) than those of the model group. These
findings suggest that succinyl rotundic acid increases levels of Nrf2 in liver
tissue of rats with alcoholic liver injury, increasing the amount of nuclear
transfer, further activating the expression of downstream antioxidant proteins,
and enhancing the oxidation defense system so as to protect the liver.
Table 1: Body weights and body weight increase of rats in various
groups (x ± s)
|
Group |
n |
Body weight |
Weight
increase |
|
|
Before
experiment |
After
experiment |
|||
|
12 |
193.56
± 4.78 |
258.31
± 5.96 |
64.75 ±
11.25 |
|
|
10 |
195.24
± 5.26 |
225.15
± 7.82 |
29.91 ±
10.32 |
|
|
10 |
196.23
± 4.32 |
245.89
± 6.15 |
49.66 ±
9.68 |
|
|
11 |
195.34
± 3.18 |
244.12
± 5.93 |
48.78 ±
10.48 |
|
|
11 |
196.54
± 5.69 |
245.79
± 6.48 |
49.25 ±
11.24 |
|
Table 2: Levels of serum LDH, AST, ALT, NOS, NO in alcohol liver
injury of rats (x ± s)
|
Groups |
LDH (U/L) |
AST (U/L) |
ALT (U/L) |
NOS (U/mL) |
NO (µmol/L) |
|
1015.26
± 105.48 |
105.46
± 17.28 |
32.78 ±
5.29 |
19.56 ±
3.48 |
20.47 ±
3.47 |
|
|
1534.15
± 114.25** |
157.68
± 24.31* |
45.37 ±
4.89 |
34.75 ±
2.09*** |
35.28 ±
2.08*** |
|
|
1089.24
± 123.14&& |
101.57
± 12.36& |
31.36 ±
5.19& |
28.47 ±
2.58# |
30.09 ±
1.48## |
|
|
947.26
± 108.97&&& |
93.27 ±
15.68&& |
29.48 ±
3.48& |
25.67 ±
2.08& |
28.32 ±
2.05#& |
|
|
908.46
± 98.56&&& |
87.68 ±
17.29&& |
26.18 ±
5.17&& |
22.34 ±
2.81&& |
25.79 ±
2.36&& |
Note: *Blank control vs.
Model control; #Blank control vs.
dose groups; &Model control vs. dose groups
Table 3: Levels of hepatic MDA, GSH, TG in alcohol liver injury
of rats (x ± s)
|
Groups |
MDA (nmol/mg) |
GSH
(mmol/mg) |
TG
(mmol/g) |
|
Blank
control |
0.62 ±
0.11 |
21.15 ±
2.45 |
0.31 ±
0.02 |
|
Model
control |
1.42 ±
0.15*** |
11.34 ±
1.78** |
0.48 ±
0.08* |
|
Low
dose |
1.24 ±
0.23## |
13.52 ±
1.63# |
0.44 ±
0.05 |
|
1.01 ±
0.10& |
15.69 ±
2.78 |
0.38 ±
0.06 |
|
|
High
dose |
0.85 ±
0.09&& |
18.45 ±
2.05& |
0.33 ±
0.04& |
*Blank control vs. Model control; #Blank
control vs. dose groups; &Model
control vs. dose groups
Levels of HO-1 in liver tissue in the model group were
significantly lower (P < 0.001)
than those of the normal group. The high- and middle-dose groups showed
significantly greater levels of HO-1 protein (P < 0.01 and P <
0.05, respectively) (Fig. 4C–D) than those of the model group. These findings
suggest that succinyl %20730-734_files/image002.png)
Fig. 2: Effects
of SRA on ALT, NOS and NO levels in serum. Values are expressed as means ± SEM.
Compared with normal group: ***P
< 0.001; compared with model group: #P < 0.05, ##P <
0.01
rotundic acid
enhances anti-oxidation and anti-apoptosis effects, improving cell survival in
liver tissue, and enhancing the oxidation defense system by up-regulating the
expression of antioxidant protein HO-1 in livers of rats with alcoholic
hepatitis.
Levels of IKBA in the model group were significantly lower (P < 0.001) than those of the normal
group. The high- and middle-dose group showed significantly greater levels of
IKBA than those of the normal group (P <
0.01 and P < 0.05, respectively) (Fig.
4C–D). These findings suggest that succinyl rotundic acid inhibits the
degradation of IKBA protein in the livers of rats with alcoholic liver injury
and reduces the release of NF-KB, thereby inhibiting inflammation. %20730-734_files/image004.png)
Fig. 1: Body
weight changes of SRA on SD rats and effects of SRA on LDH and AST levels in
serum. Values are expressed as means ± SEM. Compared with normal group: *P < 0.05, **P < 0.01; compared with model group: #P < 0.05, ##P < 0.01, ###P < 0.001
%20730-734_files/image006.png)
Fig. 3: Effects
of SRA on MDA, GSH and TG levels in serum. Values are expressed as means ± SEM.
Compared with normal group: *P
< 0.05, **P <
0.01, ***P < 0.001;
compared with model group: #P < 0.05, ##P
< 0.01
%20730-734_files/image008.jpg)
Fig. 4: The effect of SRA on NF-κB
and Bcl-2 signaling pathway in rat. Values
are expressed as means ± SEM. Compared with normal group: ***P < 0.001; compared with model group:
#P < 0.05, ##P < 0.01, ###P < 0.001
Levels NF-kB
p 65 protein were significantly lower than those of the normal group (P < 0.001). Compared with the model
group, the high-, middle-, and low-dose groups showed significantly lower
levels of NF-kB p 65 (P < 0.001, P < 0.001, and P < 0.05, respectively)
(Fig. 4C–D). These findings suggest that succinyl rotundic acid inhibits levels
of NF-kB p 65 protein in liver tissue of rats with alcoholic liver injury,
reducing the release of inflammatory factors, and thereby inhibiting
inflammation.
Discussion
In this study,
rats in the model group showed significant liver injury after long-term
administration of ethanol. Levels of serum LDH, AST, and NOS significantly
increased (Baghdasaryan et al. 2019; Sehgal et al. 2020;
Wu et al. 2020; Zou et al. 2020), suggesting
abnormal liver function and liver cell injury. MDA levels in liver tissue
increased significantly and those of GSH decreased, suggesting lipid
peroxidation damage in liver tissue. The increase of TG levels in liver tissue
suggests fatty degeneration of liver tissue. Succinyl rotundic acid reduced
serum levels of LDH, AST, NOS, and NO (Zhang et al. 2002; Tang et al.
2009; He et al. 2021), increasing GSH
activity, and reducing levels of MDA and TG (Jiao et
al. 2020; Noto et al. 2020), suggesting that it
ameliorates alcoholic liver injury and reduces lipid peroxidation damage and
steatosis. Levels of Bcl-2 in liver decreased while levels of Bax and caspase-3
increased (Raisova et al. 2001; Klemm et al. 2008; Mao et al. 2008). Succinyl rotundic acid inhibited
expression of CYP2E1 protein in liver of rats with alcoholic hepatitis, thereby
promoting the rapid decomposition of ethanol and reducing ethanol-induced liver
damage. By up-regulating the expression of Nrf2 protein in liver tissue with
alcoholic liver injury and increasing amounts of nuclear transfer, the
expression of downstream antioxidant proteins are further activated and the
oxidation defense system (Zhang et al. 2000; Hong et al. 2016) is
activated so as to achieve liver protection.
By up-regulating
the expression of antioxidant protein HO-1 in the liver of rats with alcoholic
hepatitis, anti-oxidative and anti-apoptotic capabilities are enhanced, cell
survival in liver tissues is improved, and the oxidation defense system is
enhanced. SRA inhibited the degradation of IKBA protein in liver tissue of rats
with alcoholic liver injury and reduced the release of NF-KB p65 (Chen et
al. 2011; Huang et al. 2017), thereby inhibiting inflammation. SRA
inhibited levels of NF-kB p 65 in alcoholic liver injury tissue and reduced the
release of inflammatory factors, thereby inhibiting inflammation.
Conclusion
Succinyl
rotundic acid reduces inflammatory reactions by reducing the levels of NOS and
NO, regulating levels of Bcl-2, Bax, and Caspase-3, and regulating NF-kB and
anti-oxidative stress pathways. It has an antagonistic effect on alcoholic
liver injury and has potential for treating clinical alcoholic liver disease.
Acknowledgements
This study
was supported by the National Natural Science Foundation of China (grant no.
31470418). Jilin Province Administration of Traditional Chinese Medicine
Project (grant no. 2020051).
Author
Contributions
Yajie Zhang:
Overall instructor; Yufang He, Fang Xia, Xu Wang: Responsible for the
experiment and operation; Lijing Li and Minlun Nan: Experimental operation
support.
References
Baghdasaryan N, G Ayvazyan, M Grigoryan, L Avetisyan, O Asatryan,
N Mnatsakanyan A Perikhanyan (2019). Liver involvement in the process of acute
respiratory infections in pediatric patients. J Infect Dev Countr 13:63S‒68S
Chen KH, BR Lin, CT Chien (2011). Emblica officinalis Gaertn.attentuates nitrosodiethylamine⁃induced apoptosis, autophagy, and inflammation in rat
livers. J Med Food 14:746‒755
Ciardullo S, E Muraca, S Perra,
E Bianconi, F Zerbini, A Oltolini, R Cannistraci, P Parmeggiani, G Manzoni, A Gastaldelli,
G Lattuada, G Perseghin (2020). Screening for non-alcoholic fatty liver disease
in type 2 diabetes using non-invasive scores and association with diabetic
complications. BMJ Open Diab
Res Care 8; Article e000904
He Z, J Chen, J Wang, L Xu, Z Zhou, M Chen, Y Zhang, M Shi
(2020). Expression of hepatitis B surface antigen in liver tissues can serve as
a predictor of prognosis for hepatitis B virus-related hepatocellular carcinoma
patients after liver resection. Eur J Gastroenterol Hepatol 33:76–82
Hong HM, MT Chen, YY Wang (2016). Research progress of liver apoptosis induced by decreasing
nitric oxide. J Pract Med 32:1533‒1535
Hsu YM, YC Hung, LH Hu, YJ Lee, MC Yin (2015).
Anti-diabetic effects of madecassic acid and rotundic acid. Nutrients 7:10065‒10075
Huang QH, LQ Xu, YH Liu, JZ Wu, X Wu, XP Lai, YC Li, ZR
Su, JN Chen, YL Xie (2017). Polydatin protects rat liver against
ethanol-induced injury: Involvement of CYP2E1/ROS/Nrf2 and TLR4/NF-kB p65
pathway. Evid Based Complem Altern Med 2017; Article 7953850
Jiao W, X Zhao, G Wu, X Zhang, H Wu, Y Cui (2020).
Bioactivation of lumiracoxib in human liver microsomes: Formation of GSH- and
amino adducts through acyl glucuronide. Drug Test Anal 12:827‒835
Klemm K, C Eipel, D Cantre, K Abshagen, MD Menger, B
Vollmar (2008). Multiple doses
of erythropoietin impair liver regeneration by increasing TNF ⁃ alpha, the Bax to Bcl⁃xL ratio and apoptotic cell death. PLoS One 3;
Article e3924
Li JJ, FF Chen, LY Song, JH Zhu, RM Yu (2017). Study
progress on antitumor activities of artemisinin and its derivatives. Chin J Biochem Pharm 37:10‒14
Li YM, JG Fan, BY Wang (2011). Guidelines for management
of alcoholic liver disease: An updated and revised edition. Mod Med Health 27:801‒804
Mao DW, YQ Chen, L Wang (2008). Relationship of Caspase⁃8 and Caspase⁃3 to apoptosis. J Liaon Univ Trad Chin Med 10:148‒150
Maryconi MJ, SC Mitchell (2014). Therapy for alcoholic
liver disease. World J Gastroenterol
20:2143‒2158
Noto D, F DiGaudio, IG Altieri, AB Cefalù, S Indelicato,
F Fayer, R Spina, C Scrimali, A Giammanco, A Mattina, S Indelicato, M Greco, D Bongiorno,
M Averna (2020). Automated untargeted stable isotope assisted lipidomics of
liver cells on high glucose shows alteration of sphingolipid kinetics. Biochim
Biophys Acta Mol Cell Biol Lipids 1865;
Article 158656
Raisova M, AM Hossini, J Eberle (2001). The Bax/Bcl ⁃ 2 ratio determines the susceptibility of human melanoma
cells to CD95/ Fas⁃mediated
apoptosis. J Invest Dermatol 117:333‒340
Sehgal R, H Singh, IP Singh (2020). Comparative study of
spironolactone and eplerenone in management of I ascites in patients of
cirrhosis of liver. Eur J Gastroenterol
Hepatol 32:535‒539
Tang Y, CB Forsyth, A Farhadi (2009). Nitric oxide mediated intestinal injury is required for
alcohol induced gut leakiness and liver damage. Alcohol Clin Exp Res
33:1220‒1230
Wei L, XL Zhang, Z He (2015). Advances in alcoholic
liver disease treatment. Chin J Clin
9:2593‒2597
Wu LH, MH Chen, JY Cai, Y Yuan, LQ Wu, HM Zhou, L Li, K Wan,
XX He (2020). The correlation between intestinal mucosal lesions and hepatic
dysfunction in patients without chronic liver disease. Medicine 99; Article e18837
Zhang GW, YY Fan, YJ Bao (2002). Nitric oxide anal liver injury. Prog Anat Sci 28:75‒78
Zhang Y, C Goodyer, A Leblanc (2000). Selective and protracted apoptosis in human primary neurons
microinjected with active caspase ⁃ 3, ⁃ 6, ⁃ 7 and ⁃ 8. J Neurosci 20:8384‒8389
Zou H, J Sun, B Wu, Y Yuan, J Gu, J Bian, X Liu, Z Liu
(2020). Effects of cadmium and/or lead on autophagy and liver injury in rats. Biol Trace Elem Res 198:206‒215