Effect Of PPARγ Agonist Rosiglitazone On The Expression Of IKKβ MRNA In The Kupffer Cells In Vitro

Objective: Nonalcoholic steatohepatitis (NASH) is an inherited- metabolic clinicopathological syndrome with histopathologic characters of hepatocyte steatosis, lobular and periportal inflammation and even focal necrosis. NASH is frequently associated with many genetic and acquired factors, such as obesity, type 2 diabetes, hyperlipidemia et al. It is estimated that the hepatic fibrosis of different degrees is detectable histologically in approximately 30% patients with NASH, so NASH becomes an important cause of cryptogenic cirrhosis. At present, the accurate underlying pathogenesis of NASH is poorly understood. According to the"two attack"theory, one of the most maturest explanatory theories about the mechanisms of NASH, multi factors are responsible for the pathological development of NASH together, for example, insulin resistance (IR), endotoxemia, oxidant stress, lipid peroxides, inflammatory cytokines activation and so on, which are all interrelated and interacted on each other.Although KCs belong to macrophage stabilized in the liver, they undertake about 80%-90% function of mononuclear phagocytic system in human. Thus, the activation of KCs plays an extremely important role in the hepatic inflammation and immunologic damage mediated by endotoxin (ET). As we know, nuclear factor kappa B (NF-κB) protein is a specific pro-inflammatory genic transcription factor that is widespread in vivo and sensitive to oxidative stress. Besides only a few of activated NF-κB plays an important physiologic function by keeping cell growth and resisting cell apoptosis, the majority of NF-κB is binding with its inhibitor, which will reside in cytoplasm as inactive dimmer in normal. But in the pathologic status, inhibit kappa B kinaseβ(IKKβ) can be activated by lipopolysaccharide (LPS) that is the principal active component of endotoxin, then phosphorylates NF-кB inhibitor (IκB) and induces disassociation of IκB/NF-кB dimer successively. By this signal pathway, abundant NF-кB is rapidly activated, and translates into nucleus to bind with its specific transcriptive sequence in DNA chain. Furthermore, it then leads to the development of NASH by promoting the expression of cytokines, adhesion molecules, inflammatory enzymes or proteins and so on.It is well known that, rosiglitazone (Ros.) is the most specific peroxisome proliferator-activated receptor gamma (PPARγ) agonist, which belongs to the insulin-sensitizing agents of thiazolidinediones (TZDs). It not only plays a key role in the regulation of glucose and lipid metabolism, but also depresses IR via the activation of the nuclear PPARγand the consequent regulation of gene transcription in the target tissue. In addition, it is demonstrated in other researches that PPARγis also widely expressed in KCs, and rosiglitazone may inhibit hepatic inflammation through suppressing the activation of NF-кB. Nevertheless, up to now, the relationship between the PPARγand IKKβin the liver is rarely reported and it is uncertain whether the anti-inflammatory role of rosiglitazone is owing to the activation of IKKβ/ IκB/ NF-кB signal pathways. In this study, to identify the inhibiting effect of rosiglitazone as PPARγspecific agonist on the signal pathways of IKKβ, we isolated and cultured hepatic inflammatory cells-KCs, which were activated by LPS and then be interfered with rosiglitazone in vitro. In this way, we observed the expressive change of IKKβmRNA in KCs and investigated molecule pathogenesis of NASH. All the evidence would indicate a new idea for treating NASH effectively.Methods: After 12 healthy male Wistar rats which were about 140±10g were fed with normal diet for a week, the KCs in the liver were isolated and purified with the methods ofⅣcollagenase digestion, density gradient centrifugalization and differential adhesion separation on the basis of hepatic portal venous cannula in situ. Subsequently the cells were counted and the viability of cells was tested by using 0.4% trypan blue solution staining, (0.5-1.0)×107 or so kupffer cells could be obtained in each liver and more than 95% cells would exclude trypan blue. Furthermore, the KCs were adjusted to the density of 2×106/L with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin and 100μg/ml streptomycin, and then were cultured in 6-well culture plate with the same medium in the humidified atmosphere at 37℃in 5% CO2. While no auto fluorescence of KCs under fluorescent microscope at 328nm wavelength was detected, one plastic dish of KCs was added into 20μl India ink (diluted to 5 percent by 1% gelatinum) and incubated for 4 hours. After the dish being rinsed with RPMI 1640 medium, the KCs'phagotrophic activity was identified by observing India ink particles swallowed by KCs. In this way, the activation and pureness of KCs were identified, which were more than 90%. The other KCs were cultured with RPMI 1640 medium at 37℃in 5% CO2 for 24 hours and then were randomly divided into 4 groups (each group had 12 wells): Group a: normal control group Group b: LPS 1μg/ml group Group c: LPS 1μg/ml+Ros. 10μmol/L group Group d: LPS 1μg/ml+Ros. 50μmol/L groupAfter KCs in each group were cultured in RPMI-1640 medium that contains different concentration of LPS and rosiglitazone for another 48 hours at 37℃in 5% CO2, the appearance and phagotrophic activity of KCs were identified under inverted phase contrast microscope. The kupffer cell- conditioned medium (KCCM) was collected and stored at -80℃for measuring the concentration of tumor necrosis factor alpha (TNFα) by enzyme linked immunosorbent assay (ELISA). The total RNA in KCs was extract with Trizol reagent and the expression of IKKβmRNA was measured using reverse transcription-polymerase chain reaction (RT-PCR).Results:1. Morphologic change of KCs under inverted phase contrast microscope: In normal group, the KCs started adhering wall after cultured for 30 minutes, the parapodiums of KCs could be observed after 12 hours, and it looked like polygonal or stellate by stretching out completely after 24 hours. The morphologic changes of KCs stimulated by LPS were more obvious in earlier time. The KCs'nucleus was bigger and the cytoplasm was more porous than that in the normal KCs. Furthermore, the capability to swallowing India ink and secreting TNFαof KCs were both promoted. However, all these changes of appearances and functions inducing by LPS can be inhibited by rosiglitazone, which would delay the stretching of KCs'shape and prolong the survival of KCs in a dose-dependent way.2. The determination of TNFαin KCCM: The levels of TNFαin KCCM in LPS 1μg/ml group (1.856±0.049) were significantly increased compared to that in normal control group (1.310±0.038 P<0.01). However, the higher concentration of TNFαstimulated by LPS were obviously decreased in the two rosiglitazone groups (1.791±0.046, 1.754±0.056 P<0.01), and the difference between the two experimental groups is significant(P<0.05).3. The expression of IKKβmRNA in KCs: The expression of IKKβmRNA was very low in KCs of the normal control group (0.224±0.014), but could be increased significantly by LPS in 1μg/ml (0.753±0.019 P<0.01). The promoting effect of LPS could be inhibited by rosiglitazone at 10, 50μmol/L (0.515±0.023, 0.410±0.021 P<0.01), and there were obviously significant difference between the two experimental groups (P<0.01). Moreover, the expression of IKKβmRNA was correlated positively with the level of TNFαin KCCM in each corresponding group (r =0.676, 0.809, 0.753, 0.626 P<0.05 or <0.01).Conclusion:1. The kupffer cells in liver of rats can be isolated and purified with the methods ofⅣcollagenase digestion, density gradient centrifugalization and differential adhesion separation, which will provide a foundation for studying the hepatic inflammation in vitro.2. The expression of IKKβmRNA can be significantly increased in KCs activated by LPS, and then promote the production and release of inflammatory mediators through the IκB / NF-κB / TNFαsignal pathway, which will play an important role in the pathogenesis of NASH.3. As the cross point of many signal transduction pathways, IKKβis responsible for the pathogenesis of NASH by leading to insulin resistant and activating inflammatory signal ways.4. PPARγspecific agonist rosiglitazone can inhibit the inflammation by depressing IR, blocking the expression of IKKβmRNA and down-regulating inflammatory mediators. All the evidence will provide a new idea for treating NASH effectively.