| Objective:Synergistic effect is an important way for the active ingredients of traditional Chinese medicine to exert their pharmacodynamic effect.In this paper,the components that can synergistically function with functional protein FGF2 in traditional Chinese medicine scutellaria baicalensis root were investigated by multi-compartment electrophoresis/mass spectrometry.Combined with FGF2,the effects of component complexes on cell proliferation,collagen secretion and cell migration of fibroblasts(NIH3T3)were investigated.And the active effect on wound healing in animal model of skin injury.Method:1.A multicompartment electrophoresis method was established to determine the parameters of the multicompartment electrophoresis separation system for FGF2,such as buffer,buffer pH,voltage,and electrophoresis time.2.The extracts of FGF2 and scutellaria baicalensis were incubated in a multi-compartment electrophoresis separation system,and the synergistic components were screened by protein electrophoresis.3.Establish lc-ms/MS method to detect the active components of scutellaria baicalensis root extract and identify the components with synergistic effect with FGF2.4.A lc-ms/MS MRM method was established for the detection of baicalin,hbaicalin and Oroxylin A-7-O-β-d-glucuronide.The three components with synergistic effect with FGF2 were quantitatively analyzed and the binding molar ratio was calculated.5.NIH3T3 cells were divided into blank control group,single standard drug administration group and protein administration group,and the proliferation of each group was detected by MTT method to determine the optimal concentration range.6.NIH3T3 cells were divided into blank control group,single-component administration group,FGF2 administration group,three-component combination administration group,FGF2 and single-component administration group,and FGF2 and three-component combination administration group.Each group was administered for 48 h according to the calculated molar ratio of components and the detected optimal concentration range.The proliferation of each group was detected by MTT method.7.NIH3T3 cells were divided into the blank control group,the FGF2 group,the three-component combined administration group and the FGF2 and three-component combined administration group for 48 hours.The collagen content of each group after administration was detected by the mouse hydroxyproline acid kit.8.NIH3T3 cells on the six-well plate were divided into a blank control group,a FGF2 group,a three-component combined administration group,and a FGF2 and three-component combined administration group.The cells in the six-well plate were given the drug for 24 hours after making the scratch model.9.A total of 72 SPF BALB/c male mice were selected and treated with 0.6cm2 round full-thickness skin tissue loss on both sides of the back,respectively,to establish a skin wound model.They were randomly divided into a blank control group,a FGF2 administration group,a FGF2 combined administration group with three components,and a three-component combined administration group.After the wound was cleaned with normal saline every d,medicine was given,and the wound area was measured and calculated by taking photos.At 4,7 and 14d,the wound tissues of each group were sampled,paraffin-embedded and sectioned,and the histopathological sections were stained by HE and Masson.Ⅱ-6,VEGF and CD31 were detected by immunoh istochem istry.Results:1.The parameters of the multi-compartment electrophoresis separation system were:the buffer solution was 10 mM ammonium acetate,the applied voltage was 14V,the time was 15 min,pH6.8,the incubation temperature was 37℃,and the migration rate of the target protein to the negative receiving chamber was 37.8%(RSD=3.14%).2.Three components with potential synergistic effect with FGF2 in the extract of scutellaria baicalensis root were screened by the multi-compartment electrophoresis separation system,and the qualitative detection was baicalin,scutellarin,melaleucin a-7-0-tan-d-glucuronidin.Its synergistic molar ratio is 3:1:1,and FGF2 is bound by hydrogen bond.3.FGF2 had the best proliferation effect at 50 ng/mL,with the proliferation rate of 162.25%(RSD=3.4%).FGF2 combined with three potential synergistic components can promote cell proliferation,with a proliferation rate of 212.79%(RSD=8.22%),increased collagen concentration,and a proliferation rate of 123.1%(RSD=5.49%),which is significantly different from that of FGF2 alone(P<0.05).It also improves the ability of cells to migrate.4.FGF2 combined with the three components had the best effect on wound healing,with a significantly reduced degree of inflammatory cell infiltration,orderly and dense collagen and fibroblasts,and a significantly increased density of new blood vessels compared with the model group.5.Combined use of FGF2 and three ingredients on the wound can down-regulate the expression of il-6,inhibit the release of inflammatory factors on the wound and reduce the inflammatory response.Up-regulation of VEGF expression promotes angiogenesis and accelerates wound healing.Up-regulation of CD3 1 expression promotes endothelial cells,angiogenesis and muscle fiber proliferation.Conclusion:Based on the separation system of multi-compartment electrophoresis in series with lc-ms/MS,three active components which can synergistically promote wound healing with FGF2 were selected from the extract of scutellaria baicalin,baicalin and Oroxylin A-7-O-β-d-glucuronide,respectively.The synergistic molar ratio was 3:1:1.FGF2 combined with three synergistic components can promote fibroblast proliferation,increase collagen concentration and accelerate cell migration.FGF2 combined with three synergistic components can promote wound healing in BALB/c mice.This paper provides a reference for the research on the synergistic action of traditional Chinese medicine and drug development. |
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