Scutellaria Barbata Inhibits Colorectal Cancer Stem Cell Self-renewal And Differentiation By Regulating Non-canonical Hedgehog Signaling Pathway

Objective:To investigate the effects of ethanol extract of Scutellaria barbata D.Don on the self-renewal and differentiation of colorectal cancer stem cells in vivo and in vitro,and to elucidate its molecular mechanism from non-canonical Hedgehog signaling pathway,and provide a new experimental basis for Scutellaria barbata anti-colorectal tumor.Methods:（1）evaluate the quality of Scutellaria barbata D.Don by high performance liquid chromatography（HPLC）with Scutellaria barbata D.Don as reference.（2）transfect colorectal tumor HT-29 cells with pLV-U6-hSMOshRNA-EGFP lentivirus to screen HT-29^Smo-cell,Colorectal cancer stem cells(HT-29^Smo-CSC)were sorted by magnetic bead antibody labeling;（3）determine the in vitro activity of stem cells by cck-8 assay.（4）The effect of Scutellaria barbata D.Don on the growth state of HT-29^Smo-CSC in vitro was studied by 3D culture method;（5）Stem cell globule experiment to study the effect of Scutellaria barbata D.Don on HT-29^Smo-CSC self-renewal in vitro;（6）exam the growth and transformation of HT-29^Smo-stem cell by soft AGAR colony experiment;（7）Study on the Differentiation of HT-29^Smo-CSC in vitro by Differentiation Experiment（8）use qRT-PCR to study the effects of Scutellaria barbata D.Don on the expression of cgenes Shh,Ptch1,Gli1,Sufu,CD133,c-Myc,Ki67,CK20,Nanog.（9）Effect of Scutellaria Barbata L.on Related Proteins Gli1,c-Myc and Shh in HT-29 ^Smo-CSC by Western Blot.（10）inoculate BALB/c nude mice with colorectal cancerHT-29^Smo-CSC,and construct subcutaneous transplanted tumor models.The tumor models were treated with drug intervention for 24 days,respectively.Study the effect of radix pennii on the growth of transplanted tumor of colorectal cancer HT-29^Smo-CSC by detecting the volume and weight of transplanted tumor.（11）use qRT-PCR method to detect the expressions of Shh,Ptch1,Gli1,Sufu,CD133,c-myc,CK20,Ki67 and Nanog genes in transplanted nude mice.（12）Use Wester Blot to detect the effect of Scutellaria barbata D.Don on the expression of Gli1,c-myc and Shh proteins in transplanted tumor in nude mice.（13）Use IHC method to detect the expression of Gli1,c-myc,ki 67 and CD133 proteins.Results:（1）the content of total flavones derived from Scutellaria barbata D.Don was1.29%and the scutellarin was 0.26%。（2）ht-29 cells transfected with lentivirus showed green fluorescence,but Smo protein was not expressed.（3）the ethanol extract from Scutellaria barbata D.Don inhibited the activity ofHT-29^Smo-CSC in vitro,and it was dose-dependent.（4）Scutellaria barbata D.Don can significantly inhibit the sphericity of HT-29^Smo-CSC in vitro.（5）Scutellaria barbata D.Don significantly inhibited the growth of HT-29^Smo-CSC cultured in 3D,and the higher the concentration,the worse the morphology;（6）the growth and transformation of HT-29 ^Smo-stem cells were significantly inhibited by Scutellaria barbata D.Don（P<0.05）.（7）Scutellaria barbata D.Don inhibits the differentiation ability of HT-29^SMO-CSC in vitro;（8）Scutellaria barbata D.Don up-regulated the expression level of Sufu gene,down-regulated the expression levels of Shh、Ptch1、Gli1,CD133,t c-myc,CK20,Ki67 and Nanog gene.（9）the Gli1,c-myc and Shh proteins expression levels were significantly down-regulated by Scutellaria barbata D.Don.（10）Scutellaria barbata D.Don.inhibited the growth of transplanted tumor of Colorectal Cancer Xenografts nude mice;（11）Scutellaria barbata D.Don significantly up-regulated the expression level of Sufu and down-regulated the expression levels of Shh,Ptch1,Gli1,CD133,c-myc,CK20,Ki67 and Nanog mRNA in the transplanted tumor.（12）WB results showed that Scutellaria barbata D.Don could decrease the expression of Gli1,c-Myc and Shh proteins in nude mice xenografts;（13）IHC results showed that Scutellaria barbata D.Don can reduce Gli1,c-Myc,Ki67 and CD133 proteins in nude mice xenografts.Conclusion:the ethanol extract from Scutellaria barbata D.Don inhibited proliferation and differentiation of HT-29^Smo-CSC,and significantly inhibited key target genes and proteins of non-canonical Hedgehog signaling pathway,indicating that Scutellaria barbata D.Don can inhibit colorectal tumor proliferation and differentiation through non-canonical Hedgehog signaling pathway.