Tetramethylpyrazine On Intervention Role Of The Vegf Signal Transduction Pathway

Objective: We researched the effect of tetramethylpyrazine(TMP) on binding of 125I-VEGF to VEGF receptor and detected the expression of KDR in human umbilical vein endothelial cells (HUVEC). To investigate the effect of tetramethylpyrazine (TMP) on the protein expression of vascular endothelial growth factor (VEGF) in human HepG2 and the effect of TMP on the mRNA expression of VEGF, in order to study the effect of TMP in VEGF signal pathing way and study the mechanism of TMP on anti-angiogenesis. To observe the effects of TMP on the proliferation of HepG2. Methods: The mice serum were collected after peritoneal injection big-dose TMP, low-dose TMP, protamine, Naltrii chlorcid. A reversed-phase high performance liquid chromatography(RP-HPLC) method was used to determine the TMP in mice serum .The culture medium of HUVEC was treated by the mice serum with different group. Radioligand binding assay(RBA) of receptor and Scatchard pot were performed to observe the changes of the maximum binding capacity(Bmax) and dissociation constant(Kd). The protein expression of KDR was detected by Western blot. HepG2 were incubated with TMP, the expression of VEGF was detected by ELISA assay, the expression of VEGF mRNA was detected by RT-PCR assay. Effects of the TMP on inhibiting proliferation of HepG2 were detected by MTT assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry(FCM). Results: Kd increased (P<0.05)and Bmax decreased(P<0.05) vs negative control showed that TMP decreased the expression and binding power of VEGFR. The low-dose TMP could not affect 125I-VEGF binding to VEGF. Protamine could inhibit 125I-VEGF binding to VEGF, Kd had no change, but Bmax decreased. The protein expression of KDR was lower than negative control. TMP decreased the expression of VEGF protein and VEGF mRNA. Big-dose TMP could inhibit HepG2 multiplication,and TMP could lead to apoptosis. Conclusion: The serum of big-dose TMP inhibit 125I-VEGF binding to VEGFR, the reduction of KDR and binding power of VEGFR might play an important role in TMP inhibiting angiogenesis. Moreover TMP might inhibit angiogenesis by decreasing VEGF and VEGF mRNA. TMP can interrupt the cell cycle and induce apoptosis. TMP might be one of drugs having a good future in preventing from the development of human liver cancer .