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Mast cell degranulation promotes ischemiaereperfusioninjury in rat liver

Mu-qing Yang, MD,a,b,1 Yuan-yuan Ma, MD,a,b,1 Shao-fu Tao, MD,a,c Jing Ding, MD,a

Long-hua Rao, MD,a Hong Jiang, MD, PhD,d,* and Ji-yu Li, MD, PhDb,*aDepartment of General Surgery, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, ChinabDepartment of General Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai, ChinacDepartment of General Surgery, Second People’s Hospital of Wuhu, Affiliated to Wannan Medical College, Anhui, ChinadDepartment of Orthopedic Surgery, Suzhou Hospital of Traditional Chinese Medicine, Jiangsu, China

a r t i c l e i n f o

Article history:

Received 13 May 2013

Received in revised form

7 August 2013

Accepted 20 August 2013

Available online 13 September 2013

Keywords:

Mast cell

Cromolyn

Compound 48/80

Degranulation

Ischemiaereperfusion injury

* Corresponding authors. Department of GenRoad, Shanghai 200072, China. Tel./fax: þ86

E-mail addresses: honghong751@126.com1 These authors contributed equally to thi

0022-4804/$ e see front matter Crown Copyhttp://dx.doi.org/10.1016/j.jss.2013.08.021

a b s t r a c t

Background: Mast cells (MCs) play a role in ischemiaereperfusion (I/R) injury in many

organs. However, a recent study found that MCs are not involved in I/R injury in isolated rat

livers that were perfused only for 1 h. The purpose of this study is to reevaluate the role of

MCs in hepatic I/R injury in rat.

Materials and methods: A warm hepatic I/R injury model of 1 h ischemia followed by 24 h of

reperfusion was used. MC modulation was induced via cromolyn injection or a method

called MC depletion using compound 48/80. The effects of MC modulation were evaluated

by toluidine blue staining and assessment of mast cell tryptase in sera. The role of MCs in

I/R injury was evaluated by hematoxylin and eosin staining graded by Suzuki criteria,

alanine aminotransferase and aspartate aminotransferase levels in sera, and malondial-

dehyde levels in liver homogenates.

Results: First, MC degranulation peaked after 2 h of reperfusion and liver damage peaked

after approximately 6 h of reperfusion. Second, a method called MC depletion previously

used in the skin with repeated injections of compound 48/80 worked similarly in the

hepatic setting. Third, stabilization of MCs with cromolyn or depletion of MCs with

compound 48/80 each decreased hepatic I/R injury. The most noticeable effects of cro-

molyn and compound 48/80 treatment were observed after approximately 6 h of

reperfusion.

Conclusions: MC degranulation promotes hepatic I/R injury in rats.

Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved.

1. Introduction a role not only in the innate immune system [2] but also in the

Mast cells (MCs) are well-characterized cells that harbor most

factors responsible for allergic disease, such as histamine and

inflammatory proteins [1]. During the past several years, our

understanding of MC biology has rapidly expanded. MCs play

eral Surgery, Shanghai T21 25076140.(H. Jiang), leejiyu@sina.c

s work.right ª 2014 Published by

adaptive immune system [3]. Furthermore, a large body of

recent evidence has shown that MCs can participate in

ischemiaereperfusion (I/R) injury in many organs, such as the

intestine [4], heart [5], and brain [6]. I/R injury is a syndrome

caused by ischemia and subsequent restoration of blood

enth People’s Hospital, Tongji University, 301 Middle Yanchang

om (J.-y. Li).

Elsevier Inc. All rights reserved.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8 171

supply to an organ [7]. Organ transplantation, shock, and

procedures to control bleeding during surgery can all lead to

I/R injury. The effects of MCs during I/R are mainly mediated

by chemicals stored within the granules of MCs that are

released by degranulation. These chemicals include hista-

mine [8], mast cell tryptase (MCT) [9], and chymase [10],

among others. These molecules can promote injury by

inducing physiological changes, such as increased tissue

leakage and leukocyte infiltration [11]. There are many

methods that can be used to study the effects of MC degran-

ulation, such as stabilizing MCs to prevent degranulation [12],

depleting MCs by degranulating most of the stored granules

[13], and using MC-deficient animals [14]. Cromolyn is a well-

known MC stabilizer. Compound 48/80, a well-known inducer

of MC degranulation, can deplete MCs through repeated

injections. The MC depletion method was previously used in

skin [13].

The role of MCs in I/R injury of the liver has not been well

characterized. A recent study [15] using an isolated perfused

rat liver model concluded that MCs are not involved in hepatic

I/R injury. However, this research was carried out in vitro and

data were collected only 1 h after reperfusion. There aremany

experimental studies on hepatic I/R injury in vivo assessed at

time point of 3 h after reperfusion or longer [16]. We therefore

sought to clarify whether MCs play a role in hepatic I/R injury

in vivo. First, we evaluated temporal changes in MC degranu-

lation and hepatic function during reperfusion after ischemia

in a warm I/R model. Second, we tested whether depleting

MCs through repeated injections of compound 48/80, as

previously used in the skin [13], could work similarly in the

liver. Finally, we examined whether MC stabilization with

cromolyn or depletion with compound 48/80 could reduce

hepatic I/R injury in vivo. We observed that depletion of MCs

with compound 48/80 to exhaust most granules before I/R

insult or stabilization of MCs with cromolyn to prevent

degranulation during I/R can protect the rat liver from I/R

injury, indicating that MC degranulation plays a role in

hepatic I/R injury.

2. Materials and methods

2.1. Ethics statement

All animal handling and experimental procedures were

approved by the Animal Care and Use Committee of the

Shanghai Jiao Tong University School of Medicine. Pentobar-

bital sodium anesthesia was used in every surgical procedure,

and efforts were made to minimize suffering as much as

possible.

2.2. Animals and reagents

All rats were purchased from Sino-British Sippr/Bk Laboratory

Animal Ltd (Shanghai, China). They were maintained in

standard conditions and fed with free water and standard

laboratory chow food ad libitum. Animals fasted for 12 h before

surgery. Cromolyn, compound 48/80, and toluidine blue were

purchased from Sigma (St Louis, MO).

2.3. Surgical procedure

A warm I/R injury model was created in rats as previously

described [17]. In brief, male Sprague-Dawley rats weighing

200e250 g were anesthetized by pentobarbital sodium (40 mg/

kg). The abdomens were opened and the liver hila were

exposed. The branches of portal veins and hepatic arteries

that enter into the left-lateral andmedian lobeswere occluded

with a nontraumatic microvascular clip for 60 min, and then

the clamp was removed to perfuse the ischemic liver lobes.

This model of partial hepatic I/R injury can avoid splanchnic

congestion and any confounding effects resulting from bowel

ischemia or hemodynamic disturbances. Reperfusion was

confirmed by an immediate color change before the abdomen

was closed. Rats without color change during reperfusion

after ischemia were excluded from furthermore analysis. The

abdomens of sham-operated rats were left open for 60 min

without I/R procedures. After closing the abdomen, 2 mL of

normal saline was injected through the penile vein to

compensate for fluid loss in each rat. During I/R, the body

temperature of rat was maintained at 37 � 0.3�C by a warm

support. After surgery, the rats were allowed to recover with

ad libitum access to food and water.

2.4. MC depletion with compound 48/80

Injection of compound 48/80 has previously been used to

deplete MCs in the skin [13]. To determine whether this

method can effectively deplete MCs in the liver, rats were

assigned to a compound 48/80eno ischemia (CMP-N) group

(n ¼ 5) and a phosphate buffered salineeno ischemia (PBS-N)

group (n ¼ 5). Briefly, a 0.1% (w/v) solution of compound 48/80

in PBS was administered to rats intraperitoneally in the

morning and evening for eight doses, starting with an evening

dose. For the first six doses, 0.6mg/kg was used, and 1.2mg/kg

was used for the last two doses. An equivalent volume of PBS

was administered intraperitoneally to control animals. At

5e6 h after the last injection, rats were euthanized directly

and samples were collected.

To determine the effects of MC depletion with compound

48/80 on hepatic I/R injury, rats (n ¼ 5) were treated as

described previously except that surgerywas performed 5e6 h

after the last injection and rats were euthanized via overdose

of pentobarbital sodium (80 mg/kg) at various time points of

reperfusion after ischemia for analysis.

2.5. MC stabilization with cromolyn

The MC stabilizer cromolyn was administered intraperitone-

ally (100 mg/kg) twice, once at 16 h and again at 40 min before

surgery. An equivalent volume of PBS was administered

intraperitoneally to control animals. Each group contained

five rats.

2.6. Sample collection

After euthanasia, blood samples were obtained from the

inferior vena cava. After coagulation, the samples were

centrifuged to collect the sera and the sera were stored

at �80�C until use. The median lobes of livers were harvested

Fig. 1 e Histologic evaluation of MC degranulationwith toluidine blue staining. MCsmainly gather around portal areas with

toluidine blueepositive granules. At different times of reperfusion after ischemia, MCs degranulate at different ratios, which

canbevisualizedbythismethod. (A)Sham; (B)1hof reperfusion; (C)2hof reperfusion; (D)4hof reperfusion; (E)6hof reperfusion;

(F) 8 h of reperfusion; (G) 12 h of reperfusion; and (H) 24 h of reperfusion. (Color version of figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8172

and fixed in 4% paraformaldehyde in PBS for histologic anal-

ysis. Left-lateral lobes were harvested, snap frozen in liquid

nitrogen, and stored at �80�C for biochemical analysis.

Fig. 2 e MC degranulation measured by toluidine blue

staining and ELISA of rat serum MCT. (A) Percentage of MC

degranulation counted by toluidine blue staining. (B) Rat

serum MCT measured by ELISA. *P < 0.05 and #P < 0.01

(compared with 2 h of reperfusion).

2.7. Histologic analysis of hepatic MC degranulation andliver damage

Specimens were fixed for 24 h, embedded in paraffin, and

sectioned at 5-mm intervals. To avoid one cell appearing in

more than one section during MC enumeration, sections to be

stained with toluidine blue were cut serially at intervals of at

least 150 mm.

For MC degranulation analysis, sections were stained with

0.5% toluidine blue in 0.5 M of hydrochloric acid (pH 0.5) for

30 min and then rinsed in distilled water. Degranulated MCs

were defined as having reduced granule density throughout

the entire cell or having at least three evident external gran-

ules [18]. The MCs were counted according to a previously

reported method [19]; namely, the number of degranulated

MCs was counted in each portal triad in high-power fields

(magnification, �400). In each rat, 20 portal triads were

examined. The ratio of MC degranulation was compared

between groups. The MC degranulation rate was defined as

the ratio of degranulatedMCs to total MCs in 20 portal triads in

each rat.

Fig. 3 e Temporal analysis of serum ALT and AST levels

after hepatic I/R injury. *P < 0.05 and #P < 0.01 (compared

with 6 h of reperfusion, respectively).

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8 173

The severity of I/R injurywas graded histologically using the

modified Suzuki criteria [20], after sections had been stained

with hematoxylin and eosin. Two sections fromeach specimen

were examined by two pathologists in a blinded manner.

2.8. MCT detection

Levels of MCT, a secretory granuleeassociated tryptic serine

protease, can be a measure of MC degranulation in rats [21],

mice [22], and humans [23]. Serum MCT levels of rats were

measured using commercially available enzyme-linked

immunosorbent assay (ELISA) kits (Boster Bio-engineering

Limited Company, Wuhan, China). The assays were per-

formed following the manufacturer’s instructions. Data were

expressed in nanomoles per milliliter.

2.9. Liver enzyme assessment

Serum alanine aminotransferase (ALT) and aspartate amino-

transferase (AST) were measured by a Hitachi 7600-120

Fig. 4 e Evaluation of MC depletion by toluidine blue staining af

group. (C) Comparison of the degree of MC degranulation betwee

figure is available online.)

automatic biochemical analyzer (Tokyo, Japan). The results

are expressed in international units per liter.

2.10. Liver tissue homogenate malondialdehyde assay

Malondialdehyde (MDA) was used as a marker to assess the

oxidationereduction status of the liver in liver homogenate.

MDA was measured by the thiobarbituric acid method. MDA

detection was carried out according to the instructions of

commercial kits (Nanjing Jiancheng Biological Institute,

Jiangsu, China). MDA concentrations were expressed in

nanomoles per milligram of protein.

2.11. Statistical analysis

All statistical analyses were carried out with the SPSS statis-

tical package (SPSS 19.0 for Windows; SPSS, Inc, Chicago, IL).

Values were expressed as themean� standard deviation. The

normality and homogeneity of the variances of data in each

group were tested with KolmogoroveSmirnov and Levene

tests, respectively. Unpaired t-tests were used to evaluate two

means of parametric data. For analyzing more than two

means, one-way analysis of variance was used along with

Dunnett posttest. Values of P < 0.05 were regarded as statis-

tically significant.

3. Results

3.1. Temporal changes in MC degranulation and hepaticfunction during reperfusion after ischemia

To investigate whether hepatic MC degranulation occurs

during reperfusion after ischemia, we examined livers at 1, 2,

4, 6, 8, 12, and 24 h of reperfusion. Toluidine blue staining

showed that MC degranulation peaked after 2 h of reperfusion

(Figs. 1 and 2A), which correlated well with the serum MCT

levels (Fig. 2B). These changes preceded the increase of ALT

and AST values, which peaked at about 6 h of reperfusion

(Fig. 3). On the basis of these observations, subsequent anal-

yses were performed at 2, 6, and 24 h after reperfusion.

ter compound 48/80 treatment. (A) PBS-N group. (B) CMP-N

n the PBS-N and CMP-N groups (#P< 0.01). (Color version of

Fig. 5 e Evaluation of MC degranulation by toluidine blue staining after cromolyn, PBS, and compound 48/80 treatment. (A,

D, G) Liver sections after 2, 6, and 24 h of reperfusion in the PBS-treated group. (B, E, H) Liver sections after 2, 6, and 24 h of

reperfusion in the cromolyn-treated group. (C, F, I) Liver sections after 2, 6, and 24 h of reperfusion in the compound 48/

80etreated group. (Color version of figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8174

3.2. Cromolyn and compound 48/80 treatment changesthe MC degranulation ratio

Beforewe could evaluate the effects ofMCmodulation on liver

injury, we had to determine whether the method previously

used to deplete MCs in skin was effective in liver. We found

that MC degranulation ratios in the CMP-N group and the

PBS-N group were 65.48� 4.80% and 2.42� 1.08%, respectively

(P < 0.01; Fig. 4), indicating that this method can also deplete

MCs in liver.

Wenext evaluated the effect of cromolyn and compound 48/

80 treatment on hepatic MC degranulation during I/R injury.

Representative toluidine blue staining in the PBS-treated group,

cromolyn-treated group, and compound 48/80etreated group

at 2, 6, and 24 h is shown in Figure 5. Quantification of the

staining (Fig. 6A) demonstrates that degranulation rates of the

cromolyn-treated group were significantly lower than those of

the PBS-treated group after 2 and 6 h of reperfusion (P < 0.01).

There was no significant difference in degranulation rates

between the cromolyn-treated group and PBS-treated group

Fig. 6 e MC degranulation measured by toluidine blue

staining and ELISA of rat serum MCT after cromolyn or

compound 48/80 treatment. (A) Degranulation rates

counted by toluidine blue staining in the PBS-treated

group, cromolyn-treated group, and compound 48/

80etreated group. (B) Serum MCT levels in rats of the PBS-

treated group, cromolyn-treated group, and compound 48/

80etreated group. #P < 0.01 (compared with PBS-treated

groups, respectively).

Fig. 7 e Evaluation of ALT (A) and AST (B) levels after MC

stabilization with cromolyn and MC depletion with

compound 48/80. *P < 0.05 and #P < 0.01 (compared with

PBS-treated groups).

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8 175

after 24 h of reperfusion. MC degranulation ratios at 6 and 24 h

of reperfusion were significantly different between the

compound 48/80etreated group and the PBS-treated group

(P < 0.01) but not at 2 h of reperfusion (P ¼ 0.326). MC recovery

from degranulation appeared slower in the compound 48/

80etreated group than that in the PBS-treated group.

SerumMCT levels trendedwith the histologic data (Fig. 6B).

After 2 h of reperfusion, MCT levels peaked in the compound

48/80etreatedgroup, cromolyn-treatedgroup, andPBS-treated

group. MCT levels in the cromolyn-treated group and com-

pound48/80etreatedgroupweresignificantly lower than those

in the PBS-treated group at this time point (P < 0.01). These

findings are consistent with the decreased MC degranulation

ratios in thecromolyn-treatedgroupsbut inconsistentwith the

MC degranulation ratios in the compound 48/80etreated

groups, because most MC granules, including MCT, were

depleted before ischemia in the compound 48/80etreated

groups.

3.3. Liver protection with cromolyn and compound 48/80

Serum ALT and AST levels are reliable parameters to reflect

liver injury. With the exception at 2 h of reperfusion in the

compound 48/80etreated group, ALT levels were signifi-

cantly lower at all other time points of reperfusion in the

compound 48/80etreated group and the cromolyn-treated

group than those in the PBS-treated group (Fig. 7A). With

the exception at 2 h of reperfusion, AST levels were signifi-

cantly lower at other two time points in the cromolyn-

treated group and the compound 48/80etreated group

than those in the PBS-treated group (Fig. 7B). The decrease in

ALT and AST levels in the cromolyn-treated group and

compound 48/80etreated group compared with the PBS-

treated group was most prominent after 6 h of reperfusion.

There were no differences in ALT and AST in the sham-

operated group among other groups.

3.4. Histologic changes

The liver function assay suggests that liver damage after 6 h of

reperfusion was significantly decreased after cromolyn or

compound 48/80 treatment. We therefore evaluated liver

damage at the cellular level using Suzuki criteria among the

three groups after 6 h of reperfusion. No significant changes

were observed in the liver tissues of the sham-operated

animals. The Suzuki scores of the PBS-treated group,

cromolyn-treated group, and compound 48/80etreated group

were 10.60 � 1.14, 6.40 � 1.34, and 5.80 � 1.48, respectively

(Fig. 8). Cromolyn and compound 48/80 treatment therefore

led to a significant reduction in Suzuki scores compared with

PBS treatment (P < 0.05).

3.5. MDA levels in liver tissue

As shown in Figure 9, MDA levels in the sham-operated

animals were similar. Likewise, MDA values after 2 h of

reperfusion did not differ significantly among the PBS-treated

group, cromolyn-treated group, and compound 48/80etreated

group (14.12� 2.37, 11.23� 2.50, and 11.90� 2.63, respectively)

(P > 0.05). However, after 6 h of reperfusion, MDA levels in the

PBS-treated group, cromolyn-treated group, and compound

48/80etreated group were 20.57 � 2.16, 11.56 � 2.86, and

11.25 � 2.83, respectively, indicating that cromolyn or

compound 48/80 treatment led to a significant reduction in

the levels of MDA (P < 0.01). After 24 h of reperfusion, MDA

levels were 15.63 � 1.57, 11.36 � 2.71, and 9.70 � 0.94 in the

PBS-treated group, cromolyn-treated group, and compound

48/80etreated groups. MDA levels in the compound 48/

80etreated group were significantly lower than those in the

PBS-treated group (P < 0.05).

4. Discussion

MCs have been shown to promote I/R injury in many organs,

mainly through their degranulation [8,12,14]. However, the

role of MCs in hepatic I/R injury is less clear. In the present

study, we used toluidine blue staining and detection of MCT in

serum by ELISA to determine the degree of hepatic MC

degranulation. There are many methods to detect MC

degranulation, such as toluidine blue staining [24], immuno-

histochemistry to detect specific granule contents [25], ELISA

Fig. 8 e Analysis of histology after MC stabilization with cromolyn and MC depletion with compound 48/80. Histologic

changes were examined with hematoxylin and eosin staining after 6 h of reperfusion in the sham-operated group (A), PBS-

treated group (B), cromolyn-treated group (C), and compound 48/80etreated group (D). (E) Suzuki criteria scores. Asterisk,

necrotic area; hollow arrow, congested sinusoid; and black arrow, vacuolization. Suzuki scores of the cromolyn-treated

group and compound 48/80etreated group were 6.40 ± 1.34 and 5.80 ± 1.48, respectively, which were significantly reduced

as compared with PBS-treated group (10.60 ± 1.14) (P < 0.05). (Color version of figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8176

to detect release of specific granule proteins into the serum,

and emission electronmicroscopy [26]. Toluidine blue staining

is themost commonly usedmethod inmany organs, including

the liver [27], but methods to quantify MC degranulation vary

Fig. 9 e Evaluation of MDA in rat liver after MC stabilization

with cromolyn and MC depletion with compound 48/80.

MDA levels were evaluated in sham-operated animals and

after 2, 6, and 24 h of reperfusion in the PBS-treated group,

cromolyn-treated group, and compound 48/80etreated

group. *P< 0.05 and #P< 0.01 (compared with PBS-treated

groups).

among different organs [18,24,26]. There is no generally

accepted method to quantify MC degranulation in the liver.

Because hepatic MCs mainly reside in periportal connective

tissue [25], some authors count the number of MCs in multiple

portal triads to avoid bias [19,28]. We counted total MCs and

degranulated MCs using these methods but observed a great

deal of variation. This may be because of the variation in the

areas of portal triads in different sections, as the number of

MCs correlates well with the portal triad area [19]. We there-

fore chose a method that calculated the percentage of degra-

nulated MCs among total MCs [26] in at least 20 portal triads,

which correlated well with rat serum MCT levels as measured

by ELISA. These findings suggest that this method may be

suitable for measurement of hepatic MC degranulation.

MC degranulation and MCT serum levels both peaked after

2 h of reperfusion, similar to previous findings in the heart

[29]. Taken together, these data suggest that the effects of MCs

on I/R injury are best observed after at least 2 h of reperfusion.

Indeed, many studies on the role of MCs in I/R injury choose

time points of 2 h of reperfusion or more [14,30e32]. Analyses

performed at very early time points after ischemia may

therefore overlook the long-term effects of MCs on I/R injury,

which may be the reason why Shibamoto et al. [15] found that

MCs were not involved in I/R injury in isolated rat livers

perfused only for 1 h.

If MC degranulation can promote I/R injury in the liver, we

reasoned that depleting MC granules as much as possible

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 7 0e1 7 8 177

before I/R or stabilizing MCs to prevent their degranulation

should protect liver function after I/R. We showed that MC

depletionwith compound 48/80 could be performed efficiently

and safely in the rat liver by a method used by Wei et al. [13],

which is more potent than other approaches [28]. Modulation

of MC activity in this manner may represent a useful tool to

study the effects of hepatic MC degranulation in vivo, although

other effects of compound 48/80 are found in vivo [33].

Our data show that both the stabilization of MCs with

cromolyn and depletion of MCs with compound 48/80 confer

protection against liver damage after I/R. These findings

establish the role of MCs in hepatic I/R injury, and indicate

that pharmacologic modulation of MC degranulation by

pretreatment with cromolyn or compound 48/80 before

ischemic insult may have the potential to prevent I/R injury

incurred during liver surgeries, such as partial resection or

transplantation.

Although cromolyn and compound 48/80 are so common-

ly used in MC degranulation studies, because the in vivo

responses to drugs are so complicated, the contribution of MC

degranulation to hepatic I/R injury needs to be furthermore

verified by other methods, such as using MC-deficient rats.

In addition, the mechanism by which MC degranulation

promotes hepatic I/R injury and the role of MCs in human

hepatic I/R injury needs furthermore investigation.

5. Conclusions

In this study, we demonstrated that MCs in rat liver could be

stabilized by cromolyn and depleted by compound 48/80, and

then lead to less chemicals excreting from MC degranulation

during I/R. As a result, I/R injury was alleviated.

Acknowledgment

The authors thank Jian Zhang and Lisong Shen for liver

function analyses and Meiping Shen for rat housing.

This work was supported by the National Natural Funds of

China (No. 81270555); “Shu Guang Scholar” Project, Shanghai

Municipal Educational Commission (No. 10SG20); the Key

Medical Project of Science and Technology Commission

Shanghai Municipality (No. 09411952500); and Research and

Innovation Project of Shanghai Municipal Education

Commission (No. 09YZ103).

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