The
protective
effect
of
baicalin
against
renal
ischemia-reperfusion injury through inhibition of
inflammation and apoptosis
Miao Lin 1*, Long Li2*, Liping Li2*, Gaurab Pokhrel1, Guisheng Qi1, Ruiming Rong1§
, Tongyu Zhu2§
1
Department of Urology, Fudan University Zhongshan Hospital, Shanghai, China
2
Shanghai Key Lab of Organ Transplantation, Shanghai, China
*These authors contributed equally to this work.
§
Corresponding author
Email addresses:
Miao Lin: linmiao@fudan.edu.cn
Long Li: ljlmjw@163.com
Lipiing Li: bat.150@hotmail.com
Ruiming Rong: rong.ruiming@zs-hospital.sh.cn
Tongyu Zhu: tyzhu@fudan.edu.cn
1
Abstract
Backgrounds: Renal ischemia-reperfusion injury (IRI) increases the rate
of acute kidney failure, delayed graft function, and early mortality of
kidney transplantation. The pathophysiology involved includes oxidative
stress, mitochondrial dysfunction, immune-mediated injury etc. The
anti-oxidation, anti-apoptosis, and anti-inflammation properties of
baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, have
been verified recently. This study aims to investigate the potential
effects of baicalin against renal IRI in rats.
Methods: Baicalin was intraperitoneally injected 30 minutes before
renal ischemia. We harvested serum and kidneys at 24 hours after
reperfusion. Renal function and histological change were assessed. The
markers of TLR2 and TLR4 signaling pathway, mitochondria stress, and
cell apoptosis were also evaluated.
Results: In compairision with the IR group, treatment with baicalin at
doses of 10 and 100 mg/kg significantly improved kidney function,
ameliorated histological injury, inhibited the pro-inflammatory response,
attenuated mitochondrial dysfunction, and limited tubular apoptosis.
There was a dramatic decrease of TLR2 , as well as TLR4, p-NF-κB, and
p-IκB proteins after baicalin pretreatment, along with decreased
caspase-3 activity and increased Bcl-2/Bax ratio.
Conclusion: Baicalin, at doses of 10 and 100 mg/kg can attenuate renal
2
ischemia-reperfusion injury through the inhibition of pro-inflammatory
response and mitochondria medicated apoptosis.
Key words: baicalin, ischemia-reperfusion, kidney, inflammation,
apoptosis
3
1. Introduction
Renal
ischemia-reperfusion
injury
(IRI),
an
pro-inflammatory
pathophysiological process, can increase the rate of acute kidney failure,
delayed graft function and early mortality of kidney transplantation [1].
Inducing factors of renal IRI include tissue oxygen starvation,
mitochondrial dysfunction, and ATP depletion etc [2, 3]. Renal injuries
result in the release of heat shock proteins, high mobility group box 1,
and other breakdown products, which subsequently bind to toll-like
receptor (TLR) 2 and 4 as endogenous ligands and active
immune-mediated injury termed damage-associated molecular patterns
(DAMPs). After TLR activation, an intracellular cascade of events occurs
resulting in the release of NF-κB from IκB, allowing NF-κB translocation
from the cytoplasm to the nucleus where it mediates the increase of
inflammatory cytokine gene expression leading to pro-inflammatory
response [4, 5]. As a target of immune-mediated injury, the kidney is
also capable of promoting pro-inflammatory response through the
up-regulation of TLR2 and TLR4 during IRI [6].
Reactive oxygen species production during reperfusion is thought to be
the main reason of uncontrolled oxidative stress, which subsequently
leads to mitochondrial dysfunction [7]. Apoptotic regulators, including
Bax, Bcl-2, and caspase-9, are also affected by the mitochondrial
dysfunction [8]. The direct loss of renal function after IRI is related to the
4
apoptosis of tubular epithelial cells (TECs), which can be induced by
increased pro-inflammatory cytokines or mitochondrial dysfunction [9].
Baicalin is a flavonoid glycoside isolated from Scutellaria baicalensis,
whose structure has already been clarified [10]. The root of Scutellaria
baicalensis is widely used as a traditional Chinese herb in the treatment
of infectious and inflammatory diseases with its anti-bacterial and
anti-inflammatory properties. As an active constituent of Scutellaria
baicalensis roots, baicalin has been proved to posess such protective
efficacies as well [11, 12]. Recent studies have confirmed a protective
effect of baicalin against various inflammatory diseases including
meningitis [13], sepsis [11], and chronic obstructive pulmonary disease
[12]. Side effects were rarely found. The protective effect of baicalin has
also been proved in IRI in various organs, including the heart [14], liver
[15], and brain [16]. Its mechanisms include anti-inflammatory,
anti-oxidative, and anti-apoptotic effects. These facts highlight the
promise of baicalin as a potential treatment for renal IRI.
Given the close association between the mechanisms of renal IRI and
baicalin protection, we postulated that baicalin may be effective in
protecting IRI of the kidney through the inhibition of inflammation and
apoptosis. To test the assumption, the model of rat renal IRI was
adopted. The goals of this study were: (1) to confirm the renal protection
of baicalin treatment during IRI; (2) to investigate the influence of
5
baicalin on TLR2 and TLR4 expression and subsequent inflammatory
response; (3) to investigate the influence of baicalin on mitochondrial
dysfunction and TECs apoptosis.
6
2. Material and methods
2.1. Experimental Animals
Male Wistar rats weighing 200–250g were used for this study. All rats
were housed in a local facility for laboratory animal care and held, fed on
stock diet, according to the local ethical guidelines. This study was
approved by the Bioethics Committee of Zhongshan Hospital, Fudan
University, Shanghai, according to generally accepted international
standards.
2.2. Renal ischemia-reperfusion model
Rats were randomly divided into five groups (n=6): (i) sham group; (ii) IR
+ saline group; (iii) IR + baicalin (1 mg/kg) group; (iv) IR + baicalin (10
mg/kg) group; (v) IR + baicalin (100
mg/kg) group. Renal
ischemia-reperfusion injury was induced by a 45 minutes clamping of left
renal arteries with right nephrectomy [17]. Rats were anesthetized
through an intraperitoneal injection of pentobarbital sodium at a dosage
of 40 mg/kg body weight. After a median abdominal incision, left renal
arteries were clamped for 45 minutes with serrefine. After clamp
removal, adequate restoration of blood flow was checked before
abdominal closure. Right kidney was removed then.
Sham-operated animals underwent the same surgical procedure without
7
clamping. Saline-treated animals received an intraperitoneal injection of
1 ml sterile NaCl 0.9% 30 minutes before renal clamping. In treatment
groups, baicalin (Sigma) diluted in sterile saline was injected
intraperitoneally 30 minutes before renal clamping. After operation, rats
were kept on a warming blanket for the next 12 hours with food and
water available. Animals were sacrificed 24 hours after surgery with over
dose of pentobarbital sodium. Blood and kidney were harvested.
2.3. Plasma biochemical analysis
Whole blood was centrifuged at 4℃ of 1600g for 25 minutes to obtain
the serum sample. Autobiochemistry instrument (Hitach 7060) was used
to measure the level of serum creatinine (Scr) and blood urea nitrogen
(BUN) in order to evaluate kidney function after surgery.
2.4. Histology
Renal tissue was fixed for 24 hours in 10% formalin and embedded in
paraffin. Hematoxylin and eosin (HE) staining was performed to assess
histological injury. HE stained sections were semi-quantitatively graded
at
200x magnification for TID (tubular dilation and interstitial
expansion with edema, inflammatory infiltrate) based on a scale of 0–3:
normal tubulointerstitium scored 0; mild TID affecting up to 25% field
scored 1; moderate TID affecting 25–50% field scored 2; and severe TID
8
exceeding 50% field scored 3. Examination was done blind by two
examiners in 12 randomly selected consecutive fields of an organ sample.
Each kidney is then scaled through its mean value [18].
2.5. Detection of apoptosis
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end
labeling (TUNEL) assay (KeyGEN) was used to detect apoptotic cells
according to the manufacturer’s instructions. Positive-control sections
were from hepatocarcinoma. Apoptotic cells were examined at 400x
magnification over 20 fields of tubular areas [19].
2.6. Caspase-3 activity assays
Relative caspase-3 activity in kidney was detected with caspase-3
colorimetric assay kit (KeyGEN) according to the manufacturer’s
instructions. Optical density (OD) values under 405nm were read and
stored with a microplate reader (Bio-Tek).
2.7. Western blot analysis
Cell lysates were prepared and then cytoplasmic protein of 20-40 μg
kidney tissue was obtained at 4℃. Equal amounts of proteins were
separated by SDS–PAGE and transferred onto PVDF membranes. The
primary antibodies were added and incubated at 37℃ for 2 hours with
9
gently shaking, including anti-TLR2 (Abcam), anti-TLR4 (Abcam),
anti-NF-κB (Cell Signaling Technology), anti-p-NF-κB (Cell Signaling
Technology), anti-IκB (Cell Signaling Technology), anti-p-IκB (Cell
Signaling Technology), anti-cleaved-caspase-3 (Cell Signaling Technology),
anti-caspase-9 (Cell Signaling Technology), anti-Bax (Cell Signaling
Technology), and anti-Bcl-2 (Cell Signaling Technology) respectively,
followed by incubation for 1 hours with peroxidase-conjugated
secondary antibodies (Jackson ImmunoResearch) at room temperature.
Immunoreactive bands were visualized using ECL system (Amersham
Pharmacia). To control for lane loading, the same membranes were also
probed with anti-β-actin (Epitomics) according to the molecular weight
of target proteins. The signals were quantified by scanning densitometry
using a Bio-Image Analysis System (Bio-Rad). The results from each
experimental group were expressed as relative integrated intensity
compared with that of control measured with the same batch.
2.8. Quantitative Real-Time PCR
Total RNA was extracted from rat kidney with TRIzol reagent (Invitrogen,
Shanghai, China) according to the manufacturer’s instructions. Total RNA
(3–5 μg) was transcribed into cDNA by Superscript II reverse
transcriptase
(Invitrogen)
and
random
primer
oligonucleotides
(Invitrogen). Gene-specific primers for rat IL-1β, IL-6, TNF-α, and GAPDH
10
were stated in our previous research [20]. Real-time quantitative PCR
was performed in Bio-Rad iCycler iQ system in combination with the
Absolute QPCR SYBR Green premix (Takara Bio Inc., Otsu, Shiga, Japan).
After a hot start (15 min at 95°C), the parameters for amplification were
as follows: 1 s at 95°C, 5 s at 60°C, and 10 s at 72°C for 45 cycles.
Expression levels normalized with GAPDH were calculated relative to the
housekeeping gene GAPDH using the 2−ΔΔCt method.
2.9. Statistical Analyses
Data is presented as mean ± SEM. Statistical analysis of the data was
performed with the two-tailed independent t-test between two groups,
and one-way analysis of variance (ANOVA) among more than three
groups, using SPSS 13.0 (SPSS Inc). P<0.05 was considered as statistically
significant.
3. Results
3.1. Baicalin attenuates renal dysfunction and ameliorates renal
histologic damage induced by IRI. Rats underwent renal IRI had a
significant increase of Scr (79.67 μmol/L) and BUN (22.37mmol/L) level
compared with sham group (18.00 μmol/L, 5.45 mmol/L). Baicalin
pretreatment protects renal function at a dose-dependent manner as
11
shown. High dose (10 mg/kg, 100 mg/kg) of baicalin was significantly
related to lower Scr and BUN level (Fig 1A & B). After IRI, tissue injuries
were obvious by HE staining (Fig 1C). These injuries include loss of brush
border, dilation of renal tubules, urinary cylinder etc. As previously
mentioned, a score system from 0-3 points was used to assess
histological injury. Baicalin treated groups received a lower score
compared with IR + saline group (Fig 1D).
3.2. Baicalin down-regulates the TLR2/4 and NF-κB signaling, and
inhibits the pro-inflammatory response afterwards. Accompanied with
the high level of pro-inflammatory cytokines (Fig 2A), the expression of
TLR2/4 were increased in kidney after 24 hours of IRI (Fig 2B). NF-κB is an
important
downstream
effector
of
TLR2/4
signaling.
The
activation/phosphorylation and nuclear translocation of NF-κB leads to
enlarged immuno-inflammatory response. Increased pro-inflammatory
cytokines, including TNF-α and IL-1β, would in turn promote the
phosphorylation of NF-κB. We evaluated the phosphorylation level of
NF-κB and IκB, in which a significant difference was observed in the IR +
saline group(Fig 2C). However, the expression of NF-κB was not affected.
After baicalin treatment, the increase of pro-inflammatory cytokines,
TLR2/4, p-NF-κB, and p- IκB were inhibited. In addition to the previous
results, an obvious increase of IκB protein, a NF-κB inhibitor, was also
12
observed. The degree of inhibition was positively related to the dosage
of baicalin.
3.3. Baicalin prevents mitochondrial dysfunction, down-regulates the
activity of caspase-3, and decreases apoptosis of TECs following IRI.
Activity of caspase-3 was significantly inhibited by baicalin administration
in kidney after IRI (Fig 3A). Baicalin pretreatment also leads to less
expression of cleaved caspase-3 than that in IR + saline group (Fig 3B).
Caspase-9, Bcl-2, and Bax proteins, which reflect mitochondria stress,
were analyzed by western blot (Fig 3C). Expression of pro-apoptotic
proteins, caspase-9 and Bax, were down-regulated, while expression of
anti-apoptotic protein, Bcl-2, was up-regulated, indicating inhibition of
mitochondria-mediated apoptosis by baicalin treatment. Higher dosage
(10 mg/kg or 100 mg/kg) was associated with greater inhibition. TUNEL
staining showed that most apoptotic cells were located in tubular areas,
very few were located in interstitium areas (Fig 3D). After baicalin
treatment at 10 mg/kg or 100 mg/kg, the number of apoptotic TECs was
significantly decreased.
13
4. Discussion
For the first time, we provide in vivo evidence of potential therapeutic
value of baicalin in renal IRI. Results show pretreatment of baicalin
inhibited pro-inflammatory response and immune-mediated injury
subject to IRI. Subsequent mitochondrial dysfunction and TECs apoptosis
were also ameliorated. Mechanisms underlying the protection may be
largely attributed to the inhibition of TLR2/4 and NF-κB signaling
pathway and the deactivation of mitochondria stress pathway.
As a widely used traditional Chinese herbal medicine, baicalin, purified
from Scutellaria baicalensis, has been studied for its many biological
functions, such as anti-tumor [21, 22] and anti-virus [23] properties. Its
anti-oxidation [24], anti-inflammation [11] and anti-apoptosis [25]
properties are newly discovered protective effects of baicalin, which
suggest the application of baicalin in other related diseases [14-16].
Researchers have tried many methods to attenuate renal IRI, including
various drugs [26, 27], endocrine hormones [28], erythropoietin [20],
and small interfering RNA [29]. Yet various disadvantages still prevent
them from clinical application. Facts suggest that it would be helpful to
clarify the influence of baicalin on kidney IRI and kidney transplantation.
Scr and BUN directly reflect the change of kidney function after IRI. We
evaluate kidney function 24 hours post-operation. Significant decrease of
14
Scr and BUN was observed with baicalin treatment in comparison with IR
+ saline group. This supports our former speculation that baicalin can
protect kidney from IRI. HE staining showed ameliorated pathological
damage of kidney after baicalin treatment, which is in accordance with
the change of kidney function. Higher dosage of baicalin results in better
protective effects than those with lower dosage.
IRI is an antigen independent inflammatory process that causes tissue
damage [30, 31]. Kidney IRI is associated with multiple factors, including
endothelial injury, leukocyte infiltration and tubular epithelial cell
activation, all of which trigger and exaggerate the inflammatory
response through innate and adaptive immune response [32, 33]. The
TLR2/4 signaling pathway is an important inflammatory cascade after IRI.
As an innate immune receptor on the cellular surface, its activation
would promote the nuclear translocation of NF-κB and subsequently
up-regulate pro-inflammatory cytokines and chemokines [34]. It has
been proved that TLR2 and TLR4 are constitutively expressed in proximal
and distal tubules, the thin limb of the loop of henle and the collecting
ducts with up-regulation in these sites after IRI [6]. Our results reconfirm
this conclusion. Obvious up-regulation of TLR2 and TLR4 proteins were
proved in kidney resident cells by western blot analysis. The negative
regulation of TLR signaling caused by baicalin has been proved in
LPS-stimulated human oral keratinocytes [35], oxygen-glucose deprived
15
rat microglial cells [36] and other ischemic organs [37]. We have
demonstrated that baicalin treatment decreases TLR2/4 expression and
down-regulates downstream activation of NF-κB signaling in the kidney
after IRI.
Detection of p-NF-κB and p-IκB are effective for the evaluation of NF-κB
activation. The NF-κB transcription factor, which is commonly inhibited
by IκB binding, is closely related to the expression of pro-inflammatory
genes [38]. Activation of NF-κB under IRI occurs via phosphorylation of
IκB followed by proteasome-mediated degradation. Subsequently, NF-κB
undergoes phosphorylation and translocates into the nucleus where it
regulates the pro-inflammatory response [39]. After baicalin treatment,
there proved to be a decrease in p-NF-κB and p-IκB, while IκB, the
inhibitor of NF-κB transcription factor, was increased. These results
suggest that baicalin inhibited IRI induces NF-κB activation through
decreased phosphorylation and decreased inhibitor degradation, but not
decreased NF-κB expression.
Pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α are found to
be closely associated with renal IRI. High circulation level of these
cytokines are considered as a marker of the severity. In our model, the
expression of these cytokines was significantly increased after IRI, which
is positively related to activation of TLR2/4 and NF-κB signaling.
The
oxidative
stress
and
pro-inflammatory
response
lead
to
16
mitochondria stress and immune-mediated injury, causing the injury of
tubules and apoptosis of TECs, which is the main target of IRI [2].
Mitochondrion is the central organelle in the intrinsic pathway of
apoptosis [40, 41]. Studies have proved its key role during renal IRI [7,
42]. Increased mitochondria stress during renal IRI up-regulates the ratio
of Bax/Bcl-2 and activates mitochondria mediated apoptosis. Increase of
this ratio will alter the mitochondrial membrane permeability, leading to
the release of cytochrome C, which forms apoptosome together with
caspase-9, subsequently activating downstream caspase cascade [9]. We
examined the expression of Bax and Bcl-2. Results (down-regulated Bax
and up-regulated Bcl-2) support that baicalin inhibits mitochondria
mediated apoptosis. We further evaluated the expression of caspase-9, a
component of apoptosome and an important indicator for mitochondria
mediated apoptosis [43]. Down-regulation of caspase-9 after baicalin
treatment again confirmed our former conclusion.
The results of TUNEL assay exhibited less apoptosis of TECs with baicalin
treatment than that in IR + saline group. Decreased apoptosis is related
to the deactivation of caspase cascade. Caspase-3 has been widely
accepted as a pivotal indicator of apoptosis during IRI [18, 44]. It is a
downstream effector in caspase cascade, and directly mediates
apoptosis when activated by various upstream signaling [45]. Analysis
showed that baicalin treatment significantly inhibited the activation and
17
activity of caspase-3, suggesting that baicalin related renal protection is
associated with inhibition of caspase-3 mediated apoptosis.
However, we must admit the existence of limitations in our study. Our
experiments investigated the influence of baicalin on inflammation and
apoptosis, but whether baicalin pretreatment directly affects oxidative
stress, the initial damage of IRI, is still unclear. There are also interactions
between mitochondrion and other cell organelles, such as endoplasmic
reticulum. The effect of baicalin against mitochondrial dysfunction could
be an indirect impact, possibly mediated by other organelles. What’s
more, the dose-dependent effects and mechanisms involved are still not
clear. In our study, 100 mg/kg baicalin treatment exerts the best
protection. However, the underlying mechanism still can not be
explained so far. Additional in vitro research of renal IRI is in progress in
our laboratory.
In summary, our data demonstrates for the first time that baicalin
pretreatment can significantly ameliorate renal function, reduce
pro-inflammatory cytokines, and inhibit TECs apoptosis through the
inhibition of TLR2/4 signaling pathway and mitochondria mediated cell
apoptosis pathway after renal IRI.
Authors’ Contributions
18
Miao Lin, Long Li and Liping Li was responsible for performing the
experiments, analyzing the data and drafting the manuscript. Gaurab
Pokhrel and Guisheng Qi established animal models. Ruiming Rong and
Tongyu Zhu designed the study, supervised the whole study and revised
the manuscript. All authors have read and approved the final
manuscript.
Acknowledgements
This study was supported by following fundings: National Nature Science
Foundation of China 81070595, 81170695, 81100533, 81100524; Science
and Technology Commission of Shanghai Municipality 09411952000,
10DZ2212000; Foundation of Ministry of Health of the People’s Republic
of China IHECC07-001; Special Funds of 211 works of Fudan University
211Med-XZZD02.
Conflict of interest:
The authors declare no conflict of interest.
References
1.
Kosieradzki M, Rowinski W: Ischemia/Reperfusion Injury in Kidney Transplantation:
Mechanisms and Prevention. Transplantation Proceedings 2008, 40(10):3279-3288.
2.
Eltzschig HK, Eckle T: Ischemia and reperfusion--from mechanism to translation. Nat Med
2011, 17(11):1391-1401.
3.
Brooks C, Wei Q, Cho SG, Dong Z: Regulation of mitochondrial dynamics in acute kidney
injury in cell culture and rodent models. Journal of Clinical Investigation 2009,
119(5):1275-1285.
19
4.
Liew FY, Xu D, Brint EK, O'Neill LA: Negative regulation of toll-like receptor-mediated
immune responses. Nat Rev Immunol 2005, 5(6):446-458.
5.
Wu HL, Chen G, Wyburn KR, Yin JL, Bertolino P, Eris JM, Alexander SI, Sharland AF, Chadban SJ:
TLR4 activation mediates kidney ischemia/reperfusion injury. Journal of Clinical
Investigation 2007, 117(10):2847-2859.
6.
Wolfs TG, Buurman WA, van Schadewijk A, de Vries B, Daemen MA, Hiemstra PS, van 't Veer C:
In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and
TNF-alpha mediated up-regulation during inflammation. J Immunol 2002, 168(3):1286-1293.
7.
Plotnikov EY, Kazachenko AV, Vyssokikh MY, Vasileva AK, Tcvirkun DV, Isaev NK, Kirpatovsky VI,
Zorov DB: The role of mitochondria in oxidative and nitrosative stress during
ischemia/reperfusion in the rat kidney. Kidney Int 2007, 72(12):1493-1502.
8.
Martinou JC, Youle RJ: Mitochondria in apoptosis: Bcl-2 family members and mitochondrial
dynamics. Dev Cell 2011, 21(1):92-101.
9.
Brooks C, Wei Q, Cho SG, Dong Z: Regulation of mitochondrial dynamics in acute kidney
injury in cell culture and rodent models. J Clin Invest 2009, 119(5):1275-1285.
10.
Cao Y, Mao X, Sun C, Zheng P, Gao J, Wang X, Min D, Sun H, Xie N, Cai J: Baicalin attenuates
global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic
pathways. Brain Res Bull 2011, 85(6):396-402.
11.
Zhu J, Wang J, Sheng Y, Zou Y, Bo L, Wang F, Lou J, Fan X, Bao R, Wu Y et al: Baicalin improves
survival in a murine model of polymicrobial sepsis via suppressing inflammatory response
and lymphocyte apoptosis. PLoS ONE 2012, 7(5):e35523.
12.
Li L, Bao H, Wu J, Duan X, Liu B, Sun J, Gong W, Lv Y, Zhang H, Luo Q et al: Baicalin is
anti-inflammatory in cigarette smoke-induced inflammatory models in vivo and in vitro: A
possible role for HDAC2 activity. Int Immunopharmacol 2012, 13(1):15-22.
13.
Tang YJ, Zhou FW, Luo ZQ, Li XZ, Yan HM, Wang MJ, Huang FR, Yue SJ: Multiple therapeutic
effects of adjunctive baicalin therapy in experimental bacterial meningitis. Inflammation
2010, 33(3):180-188.
14.
Chang WT, Shao ZH, Yin JJ, Mehendale S, Wang CZ, Qin Y, Li J, Chen WJ, Chien CT, Becker LB et
al: Comparative effects of flavonoids on oxidant scavenging and ischemia-reperfusion
injury in cardiomyocytes. Eur J Pharmacol 2007, 566(1-3):58-66.
15.
Kim SJ, Moon YJ, Lee SM: Protective effects of baicalin against ischemia/reperfusion injury
in rat liver. J Nat Prod 2010, 73(12):2003-2008.
16.
Cheng O, Li Z, Han Y, Jiang Q, Yan Y, Cheng K: Baicalin improved the spatial learning ability of
global ischemia/reperfusion rats by reducing hippocampal apoptosis. Brain Res 2012,
1470:111-118.
17.
Kobuchi S, Shintani T, Sugiura T, Tanaka R, Suzuki R, Tsutsui H, Fujii T, Ohkita M, Ayajiki K,
Matsumura
Y:
Renoprotective
effects
of
gamma-aminobutyric
acid
on
ischemia/reperfusion-induced renal injury in rats. Eur J Pharmacol 2009, 623(1-3):113-118.
18.
Yang B, Jain S, Ashra SY, Furness PN, Nicholson ML: Apoptosis and caspase-3 in long-term
renal ischemia/reperfusion injury in rats and divergent effects of immunosuppressants.
Transplantation 2006, 81(10):1442-1450.
19.
Furuichi K, Kokubo S, Hara A, Imamura R, Wang Q, Kitajima S, Toyama T, Okumura T,
Matsushima K, Suda T et al: Fas Ligand Has a Greater Impact than TNF-alpha on Apoptosis
and Inflammation in Ischemic Acute Kidney Injury. Nephron Extra 2012, 2(1):27-38.
20
20.
Hu L, Yang C, Zhao T, Xu M, Tang Q, Yang B, Rong R, Zhu T: Erythropoietin ameliorates renal
ischemia and reperfusion injury via inhibiting tubulointerstitial inflammation. The Journal
of surgical research 2012, 176(1):260-266.
21.
Takahashi H, Chen MC, Pham H, Angst E, King JC, Park J, Brovman EY, Ishiguro H, Harris DM,
Reber HA et al: Baicalein, a component of Scutellaria baicalensis, induces apoptosis by
Mcl-1 down-regulation in human pancreatic cancer cells. Biochim Biophys Acta 2011,
1813(8):1465-1474.
22.
Zhou QM, Wang S, Zhang H, Lu YY, Wang XF, Motoo Y, Su SB: The combination of baicalin and
baicalein enhances apoptosis via the ERK/p38 MAPK pathway in human breast cancer cells.
Acta Pharmacol Sin 2009, 30(12):1648-1658.
23.
Hu JZ, Bai L, Chen DG, Xu QT, Southerland WM: Computational investigation of the anti-HIV
activity of Chinese medicinal formula Three-Huang Powder. Interdiscip Sci 2010,
2(2):151-156.
24.
Woo
AY,
Cheng CH, Waye MM:
Baicalein
protects
rat cardiomyocytes from
hypoxia/reoxygenation damage via a prooxidant mechanism. Cardiovasc Res 2005,
65(1):244-253.
25.
Liou SF, Ke HJ, Hsu JH, Liang JC, Lin HH, Chen IJ, Yeh JL: San-Huang-Xie-Xin-Tang Prevents Rat
Hearts from Ischemia/Reperfusion-Induced Apoptosis through eNOS and MAPK Pathways.
Evid Based Complement Alternat Med 2011, 2011:915051.
26.
Mammadov E, Aridogan IA, Izol V, Acikalin A, Abat D, Tuli A, Bayazit Y: Protective Effects of
Phosphodiesterase-4-specific Inhibitor Rolipram on Acute Ischemia-reperfusion Injury in
Rat Kidney. Urology 2012.
27.
Esposito C, Grosjean F, Torreggiani M, Esposito V, Mangione F, Villa L, Sileno G, Rosso R,
Serpieri N, Molinaro M et al: Sirolimus prevents short-term renal changes induced by
ischemia-reperfusion injury in rats. Am J Nephrol 2011, 33(3):239-249.
28.
Sinanoglu O, Sezgin G, Ozturk G, Tuncdemir M, Guney S, Aksungar FB, Yener N: Melatonin
with 1,25-Dihydroxyvitamin D3 Protects against Apoptotic Ischemia-Reperfusion Injury in
the Rat Kidney. Ren Fail 2012, 34(8):1021-1026.
29.
Jia Y, Zhao Z, Xu M, Zhao T, Qiu Y, Ooi Y, Yang B, Rong R, Zhu T: Prevention of renal
ischemia-reperfusion injury by short hairpin RNA of endothelin A receptor in a rat model.
Exp Biol Med (Maywood) 2012, 237(8):894-902.
30.
Boros
P,
Bromberg
JS:
New
cellular
and
molecular
immune
pathways
in
ischemia/reperfusion injury. Am J Transplant 2006, 6(4):652-658.
31.
Halloran P, Aprile M: Factors influencing early renal function in cadaver kidney transplants.
A case-control study. Transplantation 1988, 45(1):122-127.
32.
Land WG: The role of postischemic reperfusion injury and other nonantigen-dependent
inflammatory pathways in transplantation. Transplantation 2005, 79(5):505-514.
33.
Serteser M, Koken T, Kahraman A, Yilmaz K, Akbulut G, Dilek ON: Changes in hepatic
TNF-alpha
levels,
antioxidant
status,
and
oxidation
products
after
renal
ischemia/reperfusion injury in mice. The Journal of surgical research 2002, 107(2):234-240.
34.
Wang H, Wu Y, Ojcius DM, Yang XF, Zhang C, Ding S, Lin X, Yan J: Leptospiral hemolysins
induce proinflammatory cytokines through Toll-like receptor 2-and 4-mediated JNK and
NF-kappaB signaling pathways. PLoS ONE 2012, 7(8):e42266.
35.
Luo
W,
Wang
CY,
Jin
L:
Baicalin
downregulates
Porphyromonas
gingivalis
21
lipopolysaccharide-upregulated IL-6 and IL-8 expression in human oral keratinocytes by
negative regulation of TLR signaling. PLoS ONE 2012, 7(12):e51008.
36.
Hou J, Wang J, Zhang P, Li D, Zhang C, Zhao H, Fu J, Wang B, Liu J: Baicalin attenuates
proinflammatory cytokine production in oxygen-glucose deprived challenged rat microglial
cells by inhibiting TLR4 signaling pathway. Int Immunopharmacol 2012, 14(4):749-757.
37.
Li HY, Yuan ZY, Wang YG, Wan HJ, Hu J, Chai YS, Lei F, Xing DM, Du LJ: Role of baicalin in
regulating Toll-like receptor 2/4 after ischemic neuronal injury. Chin Med J (Engl) 2012,
125(9):1586-1593.
38.
Sanz AB, Sanchez-Nino MD, Ramos AM, Moreno JA, Santamaria B, Ruiz-Ortega M, Egido J,
Ortiz A: NF-kappaB in renal inflammation. J Am Soc Nephrol 2010, 21(8):1254-1262.
39.
Diamant G, Dikstein R: Transcriptional control by NF-kappaB: elongation in focus. Biochim
Biophys Acta 2013, 1829(9):937-945.
40.
Lin CH, Chen PS, Kuo SC, Huang LJ, Gean PW, Chiu TH: The role of mitochondria-mediated
intrinsic death pathway in gingerdione derivative I6-induced neuronal apoptosis. Food
Chem Toxicol 2012, 50(3-4):1073-1081.
41.
Wang C, Youle RJ: The role of mitochondria in apoptosis*. Annu Rev Genet 2009, 43:95-118.
42.
Cruthirds DL, Novak L, Akhi KM, Sanders PW, Thompson JA, MacMillan-Crow LA:
Mitochondrial targets of oxidative stress during renal ischemia/reperfusion. Arch Biochem
Biophys 2003, 412(1):27-33.
43.
Bratton SB, Salvesen GS: Regulation of the Apaf-1-caspase-9 apoptosome. J Cell Sci 2010,
123(Pt 19):3209-3214.
44.
Yang C, Jia Y, Zhao T, Xue Y, Zhao Z, Zhang J, Wang J, Wang X, Qiu Y, Lin M et al: Naked caspase
3 small interfering RNA is effective in cold preservation but not in autotransplantation of
porcine kidneys. The Journal of surgical research 2012.
45.
Linkermann A, De Zen F, Weinberg J, Kunzendorf U, Krautwald S: Programmed necrosis in
acute kidney injury. Nephrol Dial Transplant 2012, 27(9):3412-3419.
22
Figure legends:
Figure 1
Effects of baicalin on renal function and histological change at 24 hours after renal
IRI.
Serum creatine level (A) and blood urea nitrogen (B) is significantly higher in the IR +
saline group than the sham group. Administration of 10 mg/kg and 100 mg/kg
baicalin inhibits renal dysfunction after renal IRI. Histological damage (C) after IRI is
also ameliorated by baicalin treatment according to semi-quantitative assess (D).
*p<0.05, **P<0.01, ***P<0.001
Figure 2
Effects of baicalin on IRI induced pro-inflammatory response.
IRI induced up-regulation of pro-inflammatory cytokines was inhibited by baicalin
treatment (A).
TLR2/4 and NF-κB signaling pathway were activated by IRI (B, C), which contributed
to pro-inflammatory response. Pretreatment of baicalin decreased TLR2 and TLR4
expression (B), suppressed the phosphorylation of NF-κB and IκB (C), and hence
inhibited related pro-inflammatory signaling pathway. *p<0.05, **P<0.01,
***P<0.001
Figure 3
Effects of baicalin on IRI induced mitochondria dysfunction and cell apoptosis.
the activity and expression of caspase-3 (17 kD, 12 kD), originally up-regulated by IRI,
were inhibited after baicalin treatment (A, B). We measured mitochondria stress
through caspase-9, Bax, and Bcl-2 (C). Renal IRI increased mitochondria stress, while
baicalin treatment attenuated mitochondria stress. Apoptotic cells were observed
after TUNEL assay (D). After IRI, the number of apoptotic cells was significantly
increased. Baicalin treatment at both 10 mg/kg and 100 mg/kg could significantly
decrease the number of apoptotic cells. *p<0.05, **P<0.01, ***P<0.001
23