Effects Of Scutellaria Barbata And Oldenlandia Diffusa Extracts On HBV-associated Hepatocellular Carcinoma And Its Effect On Differential Expression Of CircRNAs

Objective: To explore the anti-hepatocellular carcinoma(HCC)activity and anti-HBV activity of Scutellaria barbata,Oldenlandia diffusa,Scutellaria barbata and Oldenlandia diffusa extract in vitro and in vivo,and to probe the the possible molecular mechanism of the extract on HCC growth from the perspective of circ RNAs regulation.It may provide a theoretical basis for the study of the Scutellaria barbata and Oldenlandia diffusa extract’s the mechanism of action in the treatment of HCC.Methods: This study used a completely random experimental design types.1.The extract of Scutellaria barbata,Oldenlandia diffusa,Scutellaria barbata and Oldenlandia diffusa were prepared by heating and refluxing with ethanol,and the total flavonoids and their active ingredients luteolin and apigenin were determined.2.To clarify the effects of Scutellaria barbata and Oldenlandia diffusa extract on HBV-related Hep G2.2.15 and Hep3 B cells growth,invasion,metastasis,HBV activity,and nude mice transplanted tumor with CCK-8,plate colony formation,flow cytometry,transwell chamber,cellular immunofluorescence,as well as subcutaneous inoculation of Hep3 B xenograft model experiments in nude mice.3.This study was to preliminary detecte Scutellaria barbata and Oldenlandia diffusa extract regulating the differential expression profile of circ RNAs in Hep3 B cells by high-throughput sequencing technology,and to predicte target genes,GO functions and KEGG signaling pathways and to construct circ RNA-mi RNA-gene co-expression network diagram with bioinformatics method.Results: 1.The total flavonoids content of the extract from Scutellaria barbata,Oldenlandia diffusa,Scutellaria barbata and Oldenlandia diffusa were(5.69 ± 0.34)%,(2.47 ± 0.14)% and(5.20 ± 0.08)%,separately.The contents of luteolin were(0.143 ±0.001)%,0 % and(0.050 ± 0.001)%,and apigeninin contents were(0.072 ± 0.001)%,0 % and(0.182 ± 0.003)%,respectively.2.Compared with the control group,the Scutellaria barbata or/and Oldenlandia diffusa extract inhibited the growth of Hep G2.2.15 cell and Hep3 B cell in aconcentration-dependent and time-dependent manner within certain limits.It showed that the extract could inhibit the colony formation rate,increase the proportion of G0/G1 phase cells and decrease the G2/M phase,augment of apoptosis rate,suppress cell migration and invasion of Hep G2.2.15 and Hep3 B cells,drop the tumor volume and weight of Hep3 B tumor-bearing nude mice,reduce the expression of Ki67 and PCNA in transplanted tumors,enhance the expressions of P21 and P27,and degrade the relative expressions of HBs Ag and HBV-DNA m RNA in HCC cells,liver tissues of nude mice and serum.The differences were statistically significant(P<0.05 or P<0.01).Moreover,cell proliferation and apoptosis-related protein Cyclin D1 expression decreased slightly,P53 and Bcl-2 expression reduced,P21 and Bax expression increased(Hep3B cells P53deletion).The results of inverted microscope,transmission electron microscopy and cellular immunofluorescence showed that the morphology of HCC cells changed significantly after treatment with Scutellaria barbata and Oldenlandia diffusa extract.It manifested karyopyknosis,the number of cells decreased,the number of autophagosomes in cytoplasm increased significantly,autophagy-related proteins Beclin1 and LC3 B reduced significantly compared with the control group.3.The differential expression profiles of circ RNAs in Hep3 B cells before and after the use of the Scutellaria barbata and Oldenlandia diffusa extract were analyzed and identified.The GO function and KEGG pathway analysis also showed the biological function of differential expression of circ RNAs.A total of 5504 differential circ RNAs were bound to 751578 mi RNAs and a circ RNA-mi RNA-gene co-expression network graph was constructed by screening for HBV-associated HCC related mi RNAs.Conclusion: 1.It was confirmed that the extracts of Scutellaria barbata and Oldenlandia diffusa contained total flavonoids.The total flavonoid active ingredient of Scutellaria barbata contains luteolin and apigenin,and neither has Oldenlandia diffusa.2.The extract of Scutellaria barbata or/and Oldenlandia diffusa inhibits HCC cell proliferation,blocks the cell cycle in G0/G1 phase,induces apoptosis,supresses cell migration and invasion,depresses the expression of autophagy-related proteins,promotes the formation of autophagosome and restrains of HBs Ag and HBV-DNA replication on Hep G2.2.15 and Hep3 B cell in vitro and in vivo.However,the combination of Scutellaria barbata and Oldenlandia diffusa had no obvious synergistic effect on inhibiting the growth of HCC and HBV activity.3.Scutellaria barbata or/and Oldenlandia diffusa extract can regulate the differentialexpression of circ RNAs in Hep3 B cells,and may affect the growth of HCC cells by altering the co-expression of various circ RNA-mi RNA-genes.