| Part ?:Inhibitory effect of Clerodendrum Bungei drug-containing serum on human hepatoma MHCC97-H cells and its mechanismObjective:To investigate the effect of Clerodendrum Bungei drug-containing serum on human hepatoma MHCC97-H cells and its possible mechanism from the perspective of PI3K/Akt signaling pathway.Methods:15%drug-containing serum of Clerodendrum Bunge was applied to MHCC97-H cells,and the morphology of cells was observed under microscope;CCK8 method was used to observe the effect of drug-containing serum of Clerodendrum Bunge on cell proliferation to select the best time and concentration;AnnexinV-FITC/PI Double staining method was used to detect apoptosis;cell cycle was detected by flow cytometry;cell scratch test was used to qualitatively observe the effect of Clerodendrum Bungei drug-containing serum on migration ability of MHCC97H cells;Transwell assay was used to quantitatively detect cell invasion and migration ability;Western blot was used to detect the the expression of PTEN,p-Akt,PI3K and Akt-related proteins in key proteins in PI3K/Akt signaling pathway after 72 h of treatment;RT-PCR was used to detect the effects of Clerodendrum Bungei drug-containing serum on the expression of NF-?B,IL-2 and TNF-?mRNA after 72 h.Results:1.The results of CCK8 showed that the inhibitory effect of the Clerodendrum Bungei drug-containing serum on the proliferation of tumor cells was dose-and time-dependent.Among them,the high-dose group had the most obvious inhibitory effect,and the maximum inhibition rates at 24 h,48 h,and 72 h were 28%,32%,and 43%,respectively.2.Flow cytometry results showed that with the increase of drug-containing serum concentration,The growth of the cells was inhibited to different extents,and the percentage of apoptosis of the cells increased accordingly.The inhibitory effect of the 72 h intervention group was significant?P<0.01?.The maximum apoptosis rate of the scented peony medicated serum group was 19.48%and 19.72%.Compared with the blank group,the difference was significant?P<0.05?.3.With the increase of serum concentration,the proportion of cells in G2/M phase increased,and the proportion of cells in S phase decreased.The difference between the middle and high dose groups was significantly higher than that of the control group?P<0.05?.4.The scratch test results showed that the migration ability of MHCC97-H cells decreased after the intervention of the Clerodendrum Bungei drug-containing serum.5.Transwell test results showed that the Clerodendrum Bungei drug-containing serum significantly reduced the transmembrane cells.6.The results of Western blot showed that the expression of PTEN in different groups increased with different concentrations of Clerodendrum Bungei drug-containing serum?P<0.05?,and the expressions of PI3K and p-Akt were decreased to different degrees?P<0.05?.7.RT-PCR results show Clerodendrum Bungei drug-containing serum candecrease the expression of NF-?B,and increase the expression of TNF-?and IL-2 mRNA?P<0.05?.Conclusion:The Clerodendrum Bungei drug-containing serum has the effect of inhibiting the proliferation,migration and invasion of human hepatoma MHCC97-H cells,promoting its apoptosis and preventing its cell cycle,and its mechanism may be related to PI3K/Akt signaling pathway and regulation of immune system.Part ?:Antitumor effect and mechanism of extract of Clerodendrum Bunge on H22 tumor-bearing miceObjective:To study the inhibitory effect of Clerodendrum Bunge extract on H22 solid xenografts and its possible mechanism.Methods:A mouse H22 solid xenograft model was established by subcutaneous inoculation of H22 tumor strain.Sixty tumor-bearing mice were randomLy divided into model group,5-fluorouracil?5-Fu?group and Clerodendrum Bunge high,medium and low dose groups,12 rats in each group.Each group was given normal saline,5-Fu(25 mg·kg-1,1 time/d)and different concentrations of Clerodendrum Bunge extract intervention.After 14 days of continuous administration,the mice was sacrificed,and the tumor was removed and blood was taken from the eyeball.The body mass change,tumor inhibition rate and spleen and thymus index were detected.Peripheral blood CD4+and CD8+T lymphocytes were detected by flow cytometry.The expression levels of PTEN,PI3K,p-Akt and Akt in PI3K/Akt signaling pathway were detected by Western blot.The levels of TNF-?and IL-2 in serumwas detectedby to ELISA.Results:1.Compared with the model group,the tumor mass of the high and middle dose groups of the Clerodendrum Bunge group was significantly decreased?P<0.01?,and the thymus and spleen index increased?P<0.05?.2.In each dose group,the CD4+content in the peripheral blood of increased,CD8+content decreased,CD4+/CD8+ratio increased;3.PTEN protein expression level was up-regulated in PI3K/Akt signaling pathway,PI3K and p-Akt protein expression was decreased;4.The serum levels of TNF-?and IL-2 in the high and middle dose groups were significantly increased?P<0.05?.Conclusion:The Clerodendrum Bunge extract can inhibit the growth of H22solid xenografts,and its mechanism may be related to inhibition of PI3K/Akt signaling pathway and regulation of immune function. |
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