| Hepatic fibrosis, scarring of liver, is considered as a result of an overdone wound-healing response to various stimuli, such as alcohol consumption, hepatic virus infection and metabolic disorders, which cause chronic liver injuries, followed by inflammation response. This procedure is characterized as excessive accumulation of extracellular matrix (ECM), of which type 1 collagen (Col 1α-α) is proved to be the main constituent. Persistent fibrogenesis progressively distorts normal hepatic architecture leading to obstruction of blood circulation and impairment of liver functions, thereby giving risc to multiple complications (eg. portal hypertension, esophageal and gastric varices) and at length, driving cirrhosis and hepatocarcinoma.Upon injuries, parenchymal cells in liver produce cytokines and chemokines that facilitate Kupffer cells (KC) recruitment to the site of injuries, enhancing the release of profibrotic mediators, including cytokines and growth factors. In this process, hepatic stellate cell (HSC), a kind of mesenchymal cell, plays the pivotal role and has been proved to be the major target for fibrogenic mediators. Such activation consists of two main aspects. First, HSC is activated and transdifferentiates into myofibroblast-like cell, in which the transduction and synthesis of cytokines, inflammatory factors and ECM proteins increase dramatically. Second, the fibrogenic cell proliferates, which further promotes fibrogenesis. This procedure is precipitated by a diversity of factors, among which type beta-I Transforming growth factor (TGF-β1) and Platelet-derived growth factor (PDGF) are the most effective ones.PDGF is a dimer composed of two chains (chain A and chain B), forming three isoforms, PDGF (AA), PDGF (AB) and PDGF (BB). It is well accepted as a potent mitogen for HSCs in rat and human liver via PDGF receptor (PDGFR), and mediates various pro-fibrogenic actions, including proliferation, chemotaxis and transcription of pro-fibrogenic genes. The PDGF stimulation can induce activation of Mitogen-activated protein kinase (MAPK) signaling and the phosphatidylinositol 3-kinase (PI3K)/Akt/p70S6 kinase (p70S6K) signaling pathways, both result in HSC proliferation and collagen gene expression in vitro and in animal models. Especially, signal through sequential activation of Ras-Raf-MEK-ERK1/2 regulate cell proliferation, secretion of chemokines and also collagen synthesis.TGF-β1, highly secreted by activated KCs and epithelial cells in liver, has been conceived as a major stimulator of HSCs to produce ECM components, acting both directly and indirectly in fibrogenic remodeling processes in the liver. It not only enhances ECM proteins production, but also inhibits ECM degradation by down-regulating expression of matrix-degrading enzymes and promoting expression of matrix metalloproteinase (MMP) inhibitors. Treatment of TGF-β1 on HSC induces collagen synthesis in vitro, and in vivo over-expression of Col 1α-α results in collagen deposition and liver fibrosis. Following it binding to type Ⅱ TGF-β1 receptor, the TGF-β1/Smad pathway is activated and lead down to transduction of collagen genes and genes associated with cell transdifferentiation. Chemicals inhibiting Smad signaling have the effect of suppressing collagen gene expression and fibrogenesis. At the mean time, increased expression of TGF-β1 and both their receptors as well are detected in hepatic fibrosis。The role of Renin-angiotensin system (RAS) first came into the spotlight when it was observed that type I angiotensin receptor, or AT1 receptor, is highly expressed in HSCs after activation. The RAS was traditionally depicted as a circulating endocrine system regulating vascular resistance, sodium and water homeostasis. Many scientists believed that in liver diseases, circulating RASs function in a systemic way, as a compensatory mechanism to portal hypertension. However, accumulating studies have found that components of the RAS are abundantly expressed in many organs, like heart and kidney by myofibroblasts in the tissue repair process. And the understanding of the influence of RAS in hepatic fibrosis is gradually updated with the findings that the most important RAS mediator, AngⅡ, is generated by activated HSCs in hepatic fibrosis and studies suggested that AngⅡ could mediate and exacerbate hepatic fibrosis. AngⅡ induces both pro-inflammatory and pro-fibrotic effects in HSCs via AT1 receptor. Further evidence indicates that blockade of that RAS by utilizing angiotensin-converting enzymes inhibitor (ACEI) or AT1 receptor blockers (ARBs) impedes inflammation and ECM accumulation in chronic injury model in rats or clinical patients. Molecular researches discovered that hepatic expressed Angll may account for many physiological activities, includes cell growth, contraction, proliferation, reactive oxygen species (ROS) generation, inflammation and fibrogenesis. By inducing ROS and evoking sustained Ca2+ rise in HSCs, AngⅡ might stimulate key intracellular pathways like PI3K/Akt and MAPK that mediate proliferation and inflammatory associated gene transduction, and accentuate Smad signaling through up-regulation of TGF-β1.In fact, there exists a complicated correlation within the cytokines, growth factors and RAS which contribute to activating HSCs and accentuating fibrogenesis as a complex network. That is, the intracellular pathways mediated certain biological activities of HSC can be triggered by several mediators. For instance, both AngⅡ and PDGF activate the Ras/Raf/MEK/ERK1/2 pathway, which in turn contributes to synthesis of the mediators. And different pathways embrace profound crosstalks.During chronic liver injuries, intestinal mucosal permeability is observed lessened and giving way to an increase of bacterial translocation, resulting in the rise of LPS levels in the portal and systemic circulation. Previous studies show that LPS acts as among the strongest pro-inflammatory factors through the activation of Toll-like receptor 4 (TLR4) associated pathways, which sensitize HSCs to TGF-β1-induced signals by down-regulating the transforming growth factor TGF-β1 pseudoreceptor, Bambi.It has already been confirmed the pivotal role of AngⅡ in provoking fibrosis. In addition, in mesangial cells, monocytes and peritoneal mesothelial cells, AngⅡ has been demonstrated to up-regulate the expression of TLR4.Hitherto, the role AngⅡ takes in hepatic fibrosis and its potential relation with the inflammatory response in hepatic fibrogenesis has not yet been clarified. Hence, we were led to the hypothesis that whether Angll could up-regulate TLR4 expression in HSC as that observed in the mentioned researches, and through which enhance the signaling of LPS-TLR4 pathway. The latter, leading to the declination of Bambi expression, sensitizes the effect of TGF-β1 on HSC’s profibrotic activation, eventually driving hepatic fibrosis. Here, we report our study exploring how AngⅡ acts on TLR4 signal pathway and its profibrotic effect on HSCs. To confirm this hypothesis, we’ve designed the experiment below, which includes 4 parts:Part 1. Validate whether AngⅡ can up-regulate the expression of TLR4 in liver stellate cells at the cellular level.Gave a various density AngⅡ(0、10-8、10-7、10-6、10-5 mol/L) stimulation to T6 cells of rat liver stellate cell lines for 24 h. After the stimulation, lysed T6 cells, extracted total protein for the examination of TLR4 expression through immunoblotting. The result showed that AngⅡ-induced TLR4 expression in T6 cells presented dose-dependent effect, with a crest value of 10-6 mol/L.Part 2. Utilized Irbesartan (Irb), an AngⅡ receptor 1 (AT1) inhibitors to conform AngⅡ up-regulate HSC-expressed TLR4 by AT1 from the reverse side.The interaction between AngⅡ and AT1 was blocked by at receptor blockers. After a serum starvation for 24h, the 90%-confused T6 cells were divided into 6 groups:Control, Irb (0、10-8,10-7、10-6、10-5 mol/L). With a pretreatment of different density Irb for 1h, all Irb pretreated groups received a AngⅡ (10-6 mol/L)stimulation for 24 h. After the incentive treatment, T6 cells were harvested for TLR4 expression examination via immunoblotting. The results showed:10"’mol/L-10-3 mol/L Irb could actively suppress Ang II to up regulate TLR4 expression, accompanied with a certain dose-dependent effect.Part 3. Using qRT-PCR examining the expression of Bambi, which act as downstream effectors in LPS-TLR4 signal pathway, we investigated whether Ang II could promote the activation of LPS-TLR4 signal pathway by up-regulating TLR4 situated on the surface of HSC.Preconditioned with a serum starvation for 24 h, the 90%-confused T6 cells were divided into 4 groups:Control, Ang Ⅱ (10-6 mol/L)、LPS (100 ng/ml) and AngⅡ+LPS. Control group remained incubated for 48 h without any stimulation; Ang II group was given a stimulation of AngⅡ (10-6 mol/L) for 24 h, and remained incubated for 24 h; LPS group first sustained cultivated for 24 h, then received a LPS (100 ng/ml) stimulation for 24 h. AngⅡ+LPS group was first given a stimulation of AngⅡ (10-6 mol/L) for 24 h, later a stimulation of LPS (100 ng/ml) for 24 h. After all operations, total RNA from different groups was purified for the examination of intracellular Bambi RNA via RT-PCR. As the consequence showed, Bambi RNA in control group and Ang II group appeared unobvious differences. Bambi RNA level in LPS group was 82.7 ± 2.7% of that in control group; Bambi RNA in AngⅡ+LPS group was 71.7 ± 3.0% of that in control group, and was quantitively lower compared with LPS group. So it can be demonstrated that AngⅡ can enhance Bambi down-regulation through the LPS-TLR4 pathwayPart 4. Respectively stimulated T6 cells with AngⅡ (10-6 mol/L), LPS (100 ng/ml), TGF-β1 (300 pg/ml) in single, bigeminal and trigeminal way to investigate from intracellular level, extracellular (secretory) level and total level (the sum of both), whether AngⅡ could promote the activation of LPS-TLR4 signal pathway by up-regulating TLR4 situated on the surface of HSCPreconditioned with a serum starvation for 24 h, the 90%-confused T6 cells were divided into 8 groups:Control, AngⅡ (10-6 mol/L), LPS (100 ng/ml), TGF-β1 (300 pg/ml), AngⅡ+LPS, LPS+TGF-β1, AngⅡ+TGF-β1 and AngⅡ+LPS+TGF-β1. Control group remained incubated for 72 h without any stimulation; AngⅡ group was given a stimulation of AngⅡ (10-6 mol/L) for 24 h, and remained incubated for 48 h; LPS group first sustained cultivated for 24 h, then received a LPS (100 ng/ml) stimulation for 24 h, and then remained incubated for 24 h; TGF-β1 group first sustained cultivated for 48 h, then received a TGF-β1 (300 pg/ml) stimulation for 24 h. AngⅡ+LPS group was first given a stimulation of AngⅡ(10-6 mol/L) for 24 h, later a stimulation of LPS (100 ng/ml) for 24 h, and then remained incubated for 24 h; LPS+TGF-β1 group first sustained cultivated for 24 h, then received a LPS (100 ng/ml) stimulation for 24 h, and later received a TGF-β1 (300 pg/ml) stimulation for 24 h; AngⅡ+TGF-β1 group was first given a stimulation of AngⅡ(10-6 mol/L) for 24 h, then remained incubated for 24 h, then given a stimulation of LPS (300 ng/ml) for 24 h; AngⅡ+LPS+TGF-β1 group was first given a stimulation of AngⅡ (10-6 mol/L) for 24 h, later a stimulation of LPS (100 ng/ml) for 24 h, and at last a stimulation of TGF-β1 (300 pg/ml) for 24 h. All samples were washed with PBS for twice when the cultivating condition was changed. After the stimulation, total protein and cell culture supernatant were collected to exam intracellular and extracellular Col la expression. The result indicated:AngⅡ could promote the activation of LPS-TLR4 signal pathway by up-regulating TLR4 situated on the surface of HSC, and fortify the promoting effect caused by LPS-TLR4 pathway in hepatic fibrosis.Based on the vitro experiments mentioned above, we then investigated the differences of expression of Toll like receptor 4 (TLR4) between in normal rat liver tissue and in hepatic fibrosis (TF) tissue, as well as the influences on it exerted by Perindopril (Pe), a kind of AngⅡ converting enzyme inhibitor, and Losartan (Lo), a kind of nonpeptidic AngⅡ receptor inhibitor. Selected Wistar male rats were randomly divided into 6 groups:sham operation (Sham), choledochus ligation (BDL), Pe 14 d and 30 d and Lo 14 d and 30 d groups, with 6 rats per group. BDL group and treatment groups were operated with BDL for preparation of rat HF models, as well as given a lavage of Pe (2 mg/kg) and Lo (50 mg/kg) once a day. We detected TLR4 expression in rat livers after day 14 and day 30 respectively. From the results we found that rat liver TLR4 in BDL 14d group was (6.53 ± 1.11) (P< 0.05)times as much as in Sham group; TLR4 in Pe 14 d group and Lo 14 d group turned to be(1.71 ± 0,41) times (P> 0.05) and (0.95 ± 0.38) times (P> 0.05) than in Sham group respectively; TLR4 in Pe 30 d group and Lo 30 d group reincreased to (6.51 ± 0.87) times (P> 0.05) and (5.64 ± 0.87) times (P> 0.05) than in Sham group respectively. It showed:rat liver tissue presented a higher TLR4 expression after a HF process. Pe and Lo were capable to reduce TLR4 level in experimental HF tissue, but its suppression effect on TLR4 would decrease as a result of long-term absorption. |
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