RESUMO
ObjectiveTransforming growth factor-β1 （TGF-β1） was used to stimulate human fetal lung fibroblast 1 （HFL1） for simulating the pathological process of idiopathic pulmonary fibrosis （IPF） and thereby the effects and mechanism of medicated serum of Bupleuri Radix against IPF were investigated. MethodTGF-β1 （10 μg·L-1） was employed to stimulate HFL1， and cells were treated with medicated serum of Bupleuri Radix （5%， 10%， 15%， 20%） for 24 h. Then cell proliferation rate was determined with cell counting kit-8 （CCK-8）. Subsequently， cells were classified into the control group （20% blank serum）， TGF-β1 group （20% blank serum and 10 μg·L-1 TGF-β1）， TGF-β1 + medicated serum of Bupleuri Radix group （5% blank serum， 15% medicated serum， and 10 μg·L-1 TGF-β1）， and TGF-β1 + SIS3 group （3 μmol·L-1 SIS3， 20% blank serum， 10 μg·L-1 TGF-β1）. Based on in situ end labeling （TUNEL） staining， the apoptosis rate was examined， and mRNA expression of apoptosis-related proteins B-cell lymphoma 2 （Bcl-2）， Bcl-2 associated X protein （Bax） and myofibroblast marker α-smooth muscle actin （α-SMA） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression of α-SMA， Ras homolog enriched in brain （Rheb）， and phosphorylated （p）-Smad3 was determined by immunofluorescence. Expression of Rheb， p-Smad3， and Smad3 was examined by Western blot. ResultThe cell proliferation rate of TGF-β1 group increased compared with that of the control group （P<0.05）. The cell proliferation rate of TGF+15% medicated serum of Bupleuri Radix group and TGF+20% medicated serum of Bupleuri Radix group decreased compared with that of the TGF-β1 group （P<0.01）. Compared with the control group， TGF-β1 group showed decrease in apoptosis rate， increase in mRNA expression of Bcl-2 and α-SMA， reduction in Bax mRNA expression， and rise of α-SMA and Rheb protein expression and p-Smad3 level （P<0.05）. Compared with TGF-β1 group， TGF-β1 + medicated serum of Bupleuri Radix group and TGF-β1 + SIS3 group demonstrated high apoptosis rate， low Bcl-2 and α-SMA mRNA expression， high Bax mRNA expression， and low α-SMA and Rheb protein expression and p-Smad3 level （P<0.05）. ConclusionMedicated serum of Bupleuri Radix can inhibit TGF-β1-induced HFL1 proliferation and fibroblast-myofibroblast transition and promote fibroblast apoptosis by regulating the Smad3/Rheb axis.
RESUMO
Objective To explore whether pravastatin (Pra) inhibits mammalian target of rapamycin (mTOR) signal pathway by regulating Ras homolog enriched in brain (Rheb) protein through the comparison of gene and protein expression changes of Rheb in liver and placenta in preeclampsia (PE)-like mouse model treated with Pra. Methods C57BL/6J pregnant mice were randomly divided into two groups. The PE group was established by injecting N-nitro-L-arginine methyl ester (L-NAME) daily at gestational 7-18 days, saline was injected as contol group (Con);then giving mice Pra (PE+Pra, Con+Pra group, n=8) or normal saline (PE+N, Con+N group, n=8) every day from the 8th gestational day of pregnancy. The maternal liver and placenta tissues were collected on the 18th day of pregnancy. Western blot, real-time quantitative PCR and immunohistochemistry were used to compare the levels of Rheb protein and mRNA expression in the liver and placenta. Results (1)The results of western blot:there were no significant differences in Rheb protein expression between PE+N group (liver:0.706±0.123;placenta:0.866±0.128) and Con+N group (liver:0.732 ± 0.123; placenta: 0.909 ± 0.097), and the differences between PE+Pra group (liver: 0.669 ± 0.134;placenta:0.940 ± 0.221) and PE+N group were not significant either in liver or in placenta (all P>0.05). (2) The results of real-time quantitative PCR:when PE+N group (liver:1.026 ± 0.480;placenta:1.102 ± 0.361) compared with Con+N group (liver:1.058±0.389;placenta:1.067±0.400), PE+Pra group (liver:0.735±0.356;placenta:0.822±0.304) compared with PE+N group, there were no significant differences either in liver or in placenta (all P>0.05). (3) The results of immunohistochemistry: Rheb protein expression did not change significantly in maternal liver and placenta, there were no significant differences in protein expression levels between PE+N group and Con+N group, and between PE+Pra group and PE+N group (all P>0.05). Conclusion The inhibition of Pra on mTOR signaling pathway in some PE-like model may be independent of the expression of Rheb gene and protein.