Background: Da Huang Zhe Chong San improves the inhibitory immune status of mice with hepatocellular carcinoma by regulating Treg/Th1 balance.
Hypothesis/purpose: To study the multi-material basis and multi-mechanisms of Da Huang Zhe Chong San against hepatocellular carcinoma by regulating Treg/Th1 balance in vitro and in vivo.
Methods: UPLC-MS/MS was used to detect the dynamic changes in 29 characteristic components of different polar parts of DHZCP. H&E and TUNEL were used to check pathological condition in hepatocellular carcinoma mice. The number of CD4+T, CD8+T, Treg, Th1, and Th1-like Treg cells was counted by flow cytometry. TGF-β, IL-10, IFN-γ, and TNF-α content were detected by ELISA. α-Ketoglutarate and glutamine levels were detected by Trace1310/TSQ8000 GC-MS/MS. p-Smad2, and p-Smad3 protein levels were detected by WB, mRNA expression of Smad2, alanine-serine-cysteine transporter-2, glutaminase, and glutamate dehydrogenase were detected by RT-PCR. Simca-p multivariate data analysis software was used to evaluate the relationship between the different polar parts of Da Huang Zhe Chong San and the proportion of Treg cells.
Results: Water-soluble (PW) and ethyl acetate (PE) polar parts of Da Huang Zhe Chong San affected the HCC immune system by inhibiting the differentiation of Tregs, reversing the balance of Treg/Th1, and significantly reduced the tumor volume and weight. However, petroleum ether and n-butanol polar parts had no above actions. The changes in emodin, chrysophanol, aloe vera emodin, emodin-8-O-β-D-glycoside, gallic acid, naringenin, baicalein, wogonin, norwogonin, apigenin, chrysin, glycyrrhizin, formononetin, and palmitic acid were closely related to the changes of Treg cells, which is the main material basis of Da Huang Zhe Chong San inhibition of Treg differentiation. Additionally, PW mainly inhibit the differentiation of Treg cells by affecting the metabolism of hepatoma cells, improving tumor microenvironment acidity, and glutamine depletion. However, PE inhibited the differentiation of Treg cells mainly by regulating the TGF-β/Smad pathway.
Conclusion: In this study, accurate analysis of multi-component was combined with pharmacodynamic evaluations to identify the pharmacodynamic substances of Da Huang Zhe Chong San in regulating Treg/Th1 balance, and clarified the multi-target mechanism of Da Huang Zhe Chong San to improve tumor immunity.