Molecular Mechanisms For Tetramethylpyrazine Attenuates Sinusoidal Pathological Angiogenesis In Liver Fibrosis

BackgroundLiver fibrosis is a common pathology of various chronic liver diseases,the accurate diagnosis and treatment of liver fibrosis has been a major problem plaguing the medical community.Recent studies have shown that liver fibrosis accompanied by pathological vascular remodeling and liver sinusoidal capillaries,and played an important role in the process of treatment and prognosis of liver fibrosis.Therefore,regulation of the pathological vascular remodeling and hepatic sinusoidal capillaries were expected to become a new strategy for prevention and treatment of liver fibrosis.Our previous studies focusing on the antifibrotic potential of natural product tetramethylpyrazine(TMP),we found that TMP could inhibit the capillarization and pro-angiogenic of liver sinusoidal endothelial cells(LSECs)via block the activation of Hedgehog signaling pathway.Thus,we herein postulate that modulating Hedgehog signaling could contribute to TMP inhibition of the pathological angiogenesis in liver fibrosis.We performed experiments to testify this hypothesis.MethodsWe first evaluated the effects of TMP on liver fibrosis and pathological sinusoidal angiogenesis in rats with carbon tetrachloride(CCl4)-induced liver injury.Prevented with TMP significantly improved liver pathohistology,reduced collagen accumulation,and alleviated fibrosis.TMP(100 mg/kg)was treated by intraperitoneal injection of CCl4 to establish rat liver fibrosis model.The pathological changes of liver tissue were detected;the fenestrae of LSEC were observed by scanning electron microscopy(SEM);the levels of vascular endothelial growth factor A(VEGF-A),platelet-derived growth factor BB(PDGF-BB)and the legand of Hedgehog signaling Shh in liver homogenate and serum were detected by ELISA;the expressions of VEGF-R2,PDGF-?R,Smo and Glil at mRNA and protein levels in liver tissue were detected by real time-PCR,western blotting and immunofluorescence.Secondly,we used VEGF-A to build the primary mouse LSEC promoting angiogenesis model;the effect of TMP on pro-angiogenic ability of LSECs induced by VEGF-A was measured by Matrigel tube formation assay;the effect of prevented with TMP on expression of PDGF-BB and Shh in supernatant of LSECs induced by VEGF-A were detected by ELISA;real time-PCR,Western blotting and immunofluorescence were used to detect the effect of prevented with TMP on pro-angiogenic markers and Hedgehog signaling molecules.Finally,we further clarify the pharmacological effects of TMP on liver fibrosis by injected the Smo overexpression plasmid into the mouse tail vein.ResultsWe found that TMP can significantly reduced liver tissue damage,inhibited the deposition of collagen and thus relieve liver fibrosis in vivo.TMP can down-regulate LSEC capillarization and inhibition of pro-angiogenic markers in the fibrotic liver,suggesting a reduction of hepatic vascularization.Moreover,experiments in vitro indicated that TMP can dose-dependent inhibition of pro-angiogenic markers expressions in LSECs.In tubulogenesis assay,VEGF-A significantly promoted LSEC-mediated tube formation,but TMP treatment dose-dependently prevented LSEC-mediated tube formation.These data consistently indicated that TMP could effectively alleviate fibrosis-associated angiogenesis in fibrotic liver,and that this effect could be due to direct inhibition of LSECs.Therefore,we subsequently explored the molecular mechanisms underlying LSEC angiogenic properties.It has been cleared that LSEC as specific hepatic vascular endothelial cells can produce a variety of angiogenic molecules,of which platelet endothelial growth factor(PDGF)and angiotensin II(Ang II)plays an important role in contributing to hepatic pathological angiogenesis.On this basis,we studied the molecular mechanism of TMP in the pathological angiogenesis of LSECs.Our results showed that TMP could inhibit the pro-angiogenic markers expression in LSEC by blocking the hedgehog signaling.We found that TMP not only inhibited the levels of hedgehog ligand Shh and downregulated the expression of Ptcl and Smo two key receptors of hedgehog signaling,but also reduced the nuclear translocation of Gli1 thereby inhibited the expression of the target gene related to pro-angiogenesis.Moreover,we further used hedgehog signaling specific blockers cyclopamine(Cyc)and agonist SAG to investigate the correlation between suppressed of hedgehog signaling and inhibition of LSEC induced angiogenesis.Our results showed that Cyc can play a similarly role in suppressing of LSEC induced angiogenesis to TMP,whereas SAG reversed the effect of TMP.Taken together,these results showed that TMP the LSEC induced angiogenesis by inhibiting hedgehog signaling.Conclusions In summary,the current study indicated the molecular mechanisms and targets modulatingthe pro-angiogenic properties of LSECs,which could deepen the understanding of potential role for LSECs in the pathological angiogenesis during liver fibrosis.We also provided novel implications to reveal the molecular mechanism of TMP inhibited liver sinusoidal angiogenesis,by which points to the possibility of using TMP based anti-angiogenic drugs for the treatment of liver fibrosis.