DLPC与SAMe结合抑制肝星状细胞TIMP-1生成的研究

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"DLPC (二亚油酰磷脂酰胆碱) 和 SAMe (S-腺苷蛋氨酸) 联合使用可防止瘦素刺激的 TIMP-1(金属蛋白酶组织抑制剂-1) 生产，通过抑制 H2O2(过氧化氢)介导的信号转导途径" 在肝纤维化研究中，DLPC 和 SAMe 两种化合物因其抗氧化特性和抗纤维化作用而受到关注。H2O2 是一种在细胞信号转导过程中起到关键作用的活性氧分子，尤其在肝脏纤维化进程中的作用不容忽视。肝星状细胞 (LX-2) 是参与肝纤维化的重要细胞类型，它们在肝损伤时会活化并分泌 TIMP-1，这种蛋白质能够抑制分解纤维化组织的金属蛋白酶，从而促进纤维化的形成。这篇由 Qi Cao、Ki M. Mak 和 Charles S. Lieber 等人发表在《肝病学国际》(Liver International) 2006 年的文章，深入探讨了 DLPC 和 SAMe 如何协同作用，阻止瘦素刺激的 LX-2 细胞中 TIMP-1 的生产。瘦素是一种激素，与能量平衡和新陈代谢有关，但在肝纤维化中也可能起着促纤维化的作用。实验方法包括向 LX-2 细胞施加瘦素，并观察单独或联合应用 DLPC 和 SAMe 是否能减少 TIMP-1 的 mRNA 表达和蛋白质合成。结果显示，瘦素确实可以刺激 LX-2 细胞产生 TIMP-1，但当同时存在 DLPC 和 SAMe 时，这种刺激效应显著减弱。这表明，这两种化合物可能通过抑制 H2O2 的生成，阻断了导致 TIMP-1 生产增加的信号通路。该研究结果对于理解肝纤维化的发病机制具有重要意义，并可能为开发新的治疗策略提供线索。DLPC 和 SAMe 的抗氧化特性可能有助于保护肝细胞免受氧化应激的损害，而它们抑制 H2O2 介导的信号转导可能直接阻断了纤维化的进程。未来的研究可能进一步探索这些化合物在体内的具体作用机制以及在临床应用上的潜力，为肝纤维化患者提供新的治疗选择。

phosphorylated cytoplasmic domain at the ex-

treme terminus of OB-R

(35). Signal intensities

were quantiﬁed by imaging densitometry.

Immunoprecipitation assay for JAK phosphorylatio n

The level of JAK phosphorylation was deter-

mined at 30 min, the time at which maximal

phosphorylation by leptin occurred in LX-2 cells

(23). A 200-ml aliquot of the cell lysates was

incubated with a rabbit anti-p-JAK1 or p-JAK2

antibody (Biosource, Camarillo, CA) and then

immunoprecipitated with agarose hydrazide

beads as previously described (23). The immune

complexes (20 ml) were resolved by 12% SDS-

PAGE. Equal protein loading was controlled by

immunoblotting of the corresponding nonpho-

sphorylated JAK1 and JAK2, using the rabbit

antibodies against the respective proteins (Santa

Cruz Biotech). Signal intensities were quantiﬁed

by imaging densitometry.

Intracellular ROS and GSH determination

ROS and GSH were measured by stimulating LX-2

cells with leptin in the presence or absence of

catalase. Production of ROS was assessed by add-

ing the probe 2

-dichlorodihydroﬂuorescin diace-

tate (DCFH-DA), obtained from molecular probes

(Eugene, OR), to LX-2 culture at a ﬁnal concentra-

tion of 20 mM and incubating for 30 min in the

darkness following leptin treatment. In the cells, the

nonﬂuorescent DCFH is oxidized to the ﬂuorescent

-dichloroﬂuorescein (DCF) by ROS, mainly

peroxides, in the presence of peroxidases (36).

DCF ﬂuorescence was measured 1 h after leptin,

the time shown before at which the ﬂuorescence

peaked in LX-2 cells (23). The ﬂuorescence was

measured at 488 nm for excitation and 525 for

emission in a spectroﬂuorometer. GSH levels in

LX-2 cells were measured using the Cayman’s GSH

assay kit according to the manufacturer’s instruc-

tion. Data are reported as nmol/mg protein.

ERKI/2 and p38 MAPK phosphorylation assays

The levels of ERK1/2 and p38 phosphorylation

by leptin were determined at 2 h, the time shown

to be associated with maximal phosphorylation in

LX-2 cells (23). Phosphorylation was analyzed by

Western blots, using the components provided in

the PhosphoPlus p38 MAPK and ERKl/2

MAPK antibody kits (Cell Signaling Technology,

Beverly, MA) as previously described (23).

ERK1/2 phosphorylation was detected using a

rabbit phospho-ERK1/2 (Thr-202/Tyr-204) anti-

body and that of p38 was assayed using a rabbit

phospho-p38 (Thr-180/Tyr-182) antibody. Im-

munoblotting of total (phosphorylated and non-

phosphorylated) ERK1/2 and p38 was used as

control for equal protein loading.

Transfection of TIMP-l promoter and chloramphenicol

acetyltransferase (CAT) assay

The activity of the TIMP-1 promoter was assessed

using the CAT reporter plasmid (pBLCAT3) con-

taining nucleotides  102 to 96 (minimal promoter)

of the human TIMP-1 gene (37), which was kindly

provided by Dr. D. A. Mann of Southampton

General Hospital, Southampton, UK. Transient

transfection of LX-2 cells was performed, as pre-

viously described (23), with the LipofectAminet kit

as per the manufacturer’s protocol (Invitrogen,

Carlsbad, CA) using 320 ng of the reporter plasmid

DNA or the promoterless pBLCAT3. Transfected

cellsweretreatedwithleptinintheabsenceor

presence of catalase, SB203580, PD098059, or

DLPC1SAMe for 24 h. CAT activity was assayed

using the CAT ELISA kit and following the

manufacturer’s protocol (Roche Mol Biochem,

Indianapolis, IN). The sensitivity of the assay was

 50 pg/ml. Data are reported as ng/mg protein.

TIMP-l mRNA stability determination

To assess whether p38, ERK1/2, catalase and

DLPC1SAMe affect TIMP-l mRNA stability at

the posttranscriptional level, LX-2 cells were trea-

tedwithleptinfor24htoinduceTIMP-lmRNA.

This was followed by actinomycin D (10 mg/ml)

treatment for 20 min to block the transcription

(23). The culture medium was changed and fresh

medium containing SB203580, PD098059, cata-

lase, or DLPC1SAMe was added. After 2, 8, and

12 h of incubation, total RNA from LX-2 cells was

isolated for Northern blot analysis of TIMP-l

mRNA levels, and the decay time course in the

absence or presence of the inhibitors was analyzed.

Statistics

Data are reported as means  SEM. The signiﬁ-

cance of difference between means was analyzed

using analysis of variance followed by Student–

Newman Keuls tests. Po0.05 was considered to

be signiﬁcant.

Results

Up-regulation of TIMP-1 mRNA by leptin: effect of serum

and inhibition by different concentrations of DLPC or

SAMe

Figure 1A shows that the basal level of TIMP-1

mRNA in serum-starved LX-2 cells was compar-

223

DLPC and SAMe prevent H

and TIMP-1

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DLPC与SAMe结合抑制肝星状细胞TIMP-1生成的研究

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