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Objective To investigate the effect of angiotensin Ⅱ on protein kinase Cε (PKCε) and protein kinase Cα (PKCα) expression in hepatic stellate cells.Methods Hepatic stellate cell (HSC)-T6 cells were treated with different concentrations of angiotensin Ⅱ and the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium (MTT) assay.The expression of PKCε and PKCα was detected by immunofluorescence staining.PKCε and PKCα mRNA levels was detected by real time polymerase chain reaction (PCR).Results Angiotensin Ⅱ concentrated the proliferation of HSC-T6 cells and the level of hydroxyproline (F =25.321,13.283,P ＜ 0.001) and showed a dose-dependent effect.With the increase of angiotensin Ⅱ concentration,PKCε significantly increased and translocated in the cell membrane;PKCα increased significantly,especially in transplanted membrane and cytoplasm (F =21.387,19.431,P ＜0.01),and showed obvious dose effect.Meanwhile,Angiotensin Ⅱ increased the expression of PKCε and PKCα,and induced cell proliferation by up-regulating PKCε and PKCα mRNA levels (F =13.279,15.174,P ＜ 0.05).Conclusions Angiotensin Ⅱ can up-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner,increase the expression of protein kinase Cε and Cα,and promote the proliferation of hepatic stellate cells.
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Objective To investigate the regulation of interferon α-1b (IFNα-1b) on protein kinase Cε(PKCε) and protein kinase Cα(PKCα) which inhibit the fibrosis of hepatic stellate cells (HSC) ,and to explore its mechanism .Methods HSC-T6 cells were treated with different levels of IFNα-1b (100 , 200 ,400 ,800 and 1000 U/mL) and the proliferation of HSC-T6 cells was analyzed by methyl thiazol tetrazolium (MTT) assay .Changes of hydroxyproline level were analyzed .The expressions of PKCεand PKCαwere detected by immunofluorescence staining . PKCε, PKCα,β-catenin and Survivin mRNA levels were detected by RT-PCR . PKCε, PKCα,β-catenin and Survivin protein levels were detected by Western blot . Variance analysis was conducted by using one-way ANOVA approach . Results The inhibition rates of 100 , 200 , 400 , 800 and 1000 U/mL IFNα-1b treatment after 24 hours of administration were (15 .85 ± 1 .05)％ ,(36 .59 ± 1 .03)％ ,(45 .12 ± 1 .05)％ ,(50 .00 ± 1 .01)％ and (62 .20 ± 1 .02)％ ,respectively ,with statistically significant differences among groups (F=27 .478 , P<0 .01) .The 48h inhibition rates were (20 .87 ± 1 .09)％ ,(43 .96 ± 1 .08)％ ,(53 .85 ± 1 .08)％ ,(64 .84 ± 1 .06)％ and (74 .72 ± 1 .07)％ ,respectively ,with statistically significant differences among groups (F=25 .321 , P< 0 .01 ) . half maximal inhibitory concentration at 48 h was 343 .47 U/mL . The levels of hydroxyproline in 100 ,200 and 400 U/mL IFNα-1b groups were (7 .48 ± 0 .28) ,(6 .26 ± 0 .17) and (3 .86 ± 0 .20) μg/mL ,respectively ,which were lower than that in control group (8 .47 ± 0 .32) μg/mL .The differences were all statistically significant (t=4 .033 ,10 .564 and 21 .160 ,respective ,all P<0 .05) .The fluorescence intensities of PKCεin 100 ,200 and 400 U/mL IFNα-1b groups were all lower than that of control group .The differences were statistically significant (t=1 .984 ,2 .457 and 7 .771 ,respectively ,all P<0 .05) .The fluorescence intensities of PKCαwere also significantly lower than that of control group (t=9 .232 ,15 .921 and 22 .222 ,respectively ,all P< 0 .01) .With the increase of IFNα-1b level ,the levels of HSC-T6 PKCε,PKCα,β-catenin and survivin were significantly lower than those of control group (t=7 .020 ,24 .562 ,45 .701 and 14 .241 ,respectively ,all P<0 .01) .With the increase of IFNα-1b ,the levels of HSC-T6 PKCε,PKCα,β-catenin and survivin were significantly lower than those of control group (t=9 .564 ,4 .409 ,10 .036 and 6 .794 ,respectively ,all P<0 .01) .Conclusions IFNα-1b can down-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner ,reduce the expressions of PKCε,PKCα,β-catenin and Survivin ,and inhibit the proliferation of HSC-T6 hepatic stellate cells .
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AIM:To study the effects of N-palmitoylethanolamide ( PEA) on the anxiety-and depression-like behaviors of the mouse model induced by restraint stress , and to explore the possible mechanism of anxiolytic and antide-pressant effects of PEA .METHODS:The mice were intragastrically treated with 2.5, 5 and 10 mg/kg of PEA for 21 con-secutive days once daily .Thirty min after intragastric administration , the mice ( except the normal control group ) were placed in the glass tube to accept 4-h chronic restraint stress for 21 d.After the last administration , the mice were submit-ted to the forced stress test and the open field test (OFT) to observe the effects of PEA on the depression-like behaviors. The cumulative immobility time was recorded during the 4-min interval in the forced swimming test ( FST) or during the 5-min interval in the tail suspension test (TST).The elevated plus maze (EPM) test was used to investigate the effect of PEA on the mouse anxiety-like behaviors , and the water maze method was used to investigate the learning and memory abi-lities, spatial orientation and cognitive function of mice .After the behavior tests , the serum was collected and the hippo-campus was removed . The serum contents of adrenocorticotropic hormone ( ACTH ) , cortisol ( CORT ) and 5-hydroxytryptamine (5-HT) in the hippocampus were detected by ELISA .The changes of acetylcholinesterase ( AChE) ac-tivity in the hippocampal homogenate was measured by spectrophotometry .RESULTS:Compared with model group , in the FST or TST, the immobility time in the mice treated with PEA at 2.5~10 mg/kg and fluoxetine was significantly reduced . In the OFT, the total locomotion distance and total movement time were increased significantly in the mice , but only 10 mg/kg PEA and fluoxetine increased the numbers of rearing .In the EPM test , the percentage of the time spent in open arms, the entries into open arms and the total locomotion distance in 4 arms in the mice were significantly increased .In wa-ter maze test , PEA at 5 and 10 mg/kg and fluoxetine significantly shortened the latency to find the security zone in the mice, and PEA at 10 mg/kg and fluoxetine obviously shorten the swimming distance .Compared with model group , PEA at 10 mg/kg and fluoxetine reduced the mouse serum levels of ACTH and CORT , and the adrenal index , increased the 5-HT content and decreased the AChE activity in the hippocampus .CONCLUSION:PEA produces antagonistic effects on an-xiety-and depression-like behaviors in the mice induced by restraint stress .Its specific mechanism may be related to the re-gulation of the hypothalamic-pituitary-adrenal axis function by increasing the 5-HT level in hippocampus , thus participating in the regulation of central cholinergic system .