Expression And Effect Of IL-10/IL-10R On Hepatocytes

AIMRat hepatic fibrosis model was developed to study the variation of IL-10 expression in liver and the effect of recombination rat IL-10(rrIL-10) on rat hepatocyte. Rat primary hepatocyte was isolated to investigate the effect of IL-10 on proliferation apoptosis and IGF-1 mRNA expression of rat primary cultured hepatocytes.METHODHepatic fibrosis was indused by CCL4. 47 adult male Sprague-Dawley rats were divided into four group: normal control group, model group(+CCL4), IL-10 treated group(CCL4+IL-10) and recovered group (CCL4+no IL-10). Reverse-transcription polymerase chain reaction (RT-PCR) was used to analyze the variation of IL-10 mRNA expression . TUNEL in situ was detected to investigate the effect of IL-10 on hepatocyte apoptosis . Reverse-transcription polymerase chain reaction (RT-PCR) was used to analyze Bax/Bcl-2 mRNA from freshly isolated cells. Immunohistochemistry was performed to detect Bax/Bcl-2 protein expression in liver tissue.The liver of SD rats was perfused through portal vein with collagenase IV to isolate the hepatocytes . The primary hepatocyte was cryopreservation, resuscitation, cultured and purity accessed . IL-10/IL-10R1 mRNA and protein expression of hepatocyt were also detected . The primary cultured hepatocytes were divided into 3 groups and treated with nothing (group N), Insulin (group C), and IL-10 in combination with Insulin (group I), respectively. Nuclear cell cycle analysis, MTT, and Trypan Blue cell count, AnnexinV, AO-EB was assayed to know effect of IL-10 on hepatocytes proliferation and apoptosis . RT-PCR was assayed in IGF-1 mRNA expression of hepatocytes.RESULTRat hepatic fibrosis was developed as showed by histological examination. Real-Time PCR showed IL-10 mRNA expression of Group M was depressed contrast to Group N(P<0.01). TUNEL in situ detect showed apoptosis of Group I was significantly reduced contrast to Group R (P<0.01). Immunohistochemistry showed that hepatocytes highly expressed Bax and lowly expressed Bcl-2. Bax expression was decreased in GT contrast to GR(P<0.01). RT-PCR showed the expression of Bax/Bcl-2 mRNA was downregulated in GT compared to GR (Bax: P<0.05, Bcl-2: P<0.01).The primary hepatocyte was successfully isolated. RT-PCR showed different expression of characterization genes in primary hepatocytes group and liver tissue. RT-PCR detection found the expression of IL-10/IL-10R1 mRNA. And IL-10 protein expression was also found in primary hepatocytes through Western-blot assay. MTT analyse showed absorbance of group I was declined contrast to group C at 48h (88.41% , P<0.05). Trypan Blue cell count also showed an depression of cell quantity in group I at 48h (71.96% contrast to group C, P<0.05). Cell cycle analysis via FCM showed a decline at 24h in group I than group C and group N (59.06% and 70.18%, P<0.01). AO-EB ,AnnexinV result showed there are seldom cells of apoptosis in primary cultured hepatocytes . Semi-quantity RT-PCR showed IGF-1 mRNA expression of Group I was decreased in 24h and 48h contrast to GroupC(87.38%,89.43%), P<0.01. The differnce in 72h was none significant. CONCLUSIONPrimary hepatocytes can autocrine IL-10. IL-10 mRNA expression was decreased in hepatic fibrosis rat's liver. Rr-IL-10 depressed the apoptosis of hepatocytes in rat fibrosis liver. Cryopreservation and resuscitation causes synchronization of hepatocytes. Rr-IL-10 supressed the proliferation of primary hepatocytes and downregulated its IGF-1 mRNA expression.