ObjectiveTo study the regulatory effects of Yinchenhao Tang combined with Yinchen Zhufu Tang on the expression of regulatory T （Treg）/helper T 17 （Th17） cells cultured in vitro from the patients with hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF）. MethodsFresh peripheral blood was collected from the patients with HBV-ACLF for the separation of peripheral blood mononuclear cells （PBMCs）. Immunomagnetic beads were used to isolate primary Treg and naive CD4⁺ T cells. After in vitro expansion， naive CD4⁺ T cells were induced to differentiate into Th17 cells. Rats were treated with the clearing method （Yinchenhao Tang）， warming method （Yinchen Zhufu Tang）， and combination of clearing method with warming method （Yinchenhao Tang combined with Yinchen Zhufu Tang， also known as Wenyang Jiedu Huayu Prescription）， respectively， and then the medicated plasma samples were collected. Meanwhile， blank plasma was collected from the rats treated with normal saline. Cells were classified into blank， clearing method （5.04 g·kg^-1）， warming method （6.21 g·kg^-1）， and combination of clearing method with warming method （17.1 g·kg^-1） groups and treated with corresponding plasma. The frequency of Treg/Th17 cells was detected by flow cytometry. The level of transforming growth factor-β （TGF-β） was measured by the enzyme-linked immunosorbent assay. The cytometric bead array （CBA） was employed to measure the levels of interleukin-10 （IL-10）， interleukin-17A （IL-17A）， tumor necrosis factor-α （TNF-α）， and interleukin-23 （IL-23）. The mRNA and protein levels of Forkhead box P3 （FoxP3） and retinoic acid-related orphan receptor-gamma t （ROR-γt） were determined by Real-time PCR and Western blot， respectively. ResultsCompared with the blank group， the combination of clearing method with warming method group showed decreased frequency of Treg and Th17 cells， lowered levels of Treg cytokines （TGF-β and IL-10） and Th17 cytokines （TNF-α， IL-17A， and IL-23）， and down-regulated mRNA and protein levels of FoxP3 and ROR-γt （P<0.01）. Compared with the clearing method group， the combination of clearing method with warming method group showed decreased Treg cell frequency and down-regulated mRNA and protein levels of FoxP3. Meanwhile， the combination group showed decreased Th17 cell frequency， lowered levels of TGF-β， IL-10， TNF-α， IL-17A， and IL-23， and down-regulated mRNA and protein levels of ROR-γt （P<0.05， P<0.01）. Compared with the warming method group， the combination of clearing method with warming method group showed decreased frequency of Treg cells and down-regulated mRNA and protein levels of FoxP3. Meanwhile， the combination group showed decreased Th17 cell frequency， declined levels of TGF-β， IL-10， TNF-α， IL-17A， and IL-23， and down-regulated mRNA and protein levels of ROR-γt （P<0.05）. ConclusionThe combination of clearing method with warming method can down-regulate the expression of specific cytokines of Treg and Th17 cells， inhibit the over activation of Treg and Th17 cells， and reduce the secretion of cytokines such as TGF-β， IL-10， TNF-α， IL-17A， and IL-23， thereby alleviating inflammation and improving the prognosis of the patients with liver failure.

ObjectiveTo investigate the differential expression of intestinal flora in patients with different traditional Chinese medicine （TCM） syndrome types （Yang Huang syndrome， Yin-Yang Huang syndrome， and Yin Huang syndrome） of hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF） and clarify the biological basis of jaundice and Yin Huang syndrome in liver failure. MethodsA total of 20 cases of HBV-ACLF patients were included in the Yang Huang group， 20 cases in the Yin-Yang Huang group， 16 cases in the Yin Huang group， and 20 healthy adult volunteers. 16S rRNA gene sequencing was used to detect the diversity， species distribution， and differences of the subjects' intestinal flora， and bioinformatics analysis was conducted. ResultsCompared with those in the healthy control group， the species richness and diversity of intestinal flora in the HBV-ACLF Yang Huang group， Yin-Yang Huang group， and Yin Huang group were significantly reduced， and there were significant differences in the composition of intestinal flora compared with healthy volunteers. However， there were no significant differences in the species richness， diversity， and composition of intestinal flora among the three groups. LEfSe analysis showed that compared with the healthy control group， the HBV-ACLF Yang Huang group showed significant enrichment of Staphylococcus aureus（P<0.01）. Yin-Yang Huang group showed significant enrichment of s_Ileibacterium valens（P<0.05，P<0.01）， and the Yin Huang group showed significant enrichment of Enterococcus faecium and Streptococcus sali varius（P<0.05）. These strains may be biomarkers between the three groups of patients and the healthy control group. Compared with that in the Yin-Yang Huang group， Tyzzerella_nexilis was significantly enriched in the Yang-Huang group， and Streptococcus lactiae was significantly enriched in the Yin-Yang Huang group. Compared with that in the Yang-Huang group and the Yin-Yang Huang group， Enterococcus faecalis was significantly enriched in the Yin Huang group. The above strains may be biomarkers among the three groups of patients， and Enterococcus faecium may be a biomarker for the transition from the Yang Huang group to the Yin Huang group. ConclusionsThere are significant differences in the intestinal flora between patients with HBV-ACLF Yang Huang syndrome， Yin-Yang Huang syndrome， and Yin Huang syndrome. Enterococcus faecium is significantly enriched in the Yin Huang syndrome group， suggesting that dysbiosis of the intestinal flora may be the biological basis for jaundice and Yin Huang syndrome in liver failure.

ObjectiveTo study the expression differences of regulatory T cells （Treg） and T helper 17 cells （Th17） in the peripheral blood of patients with hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF） in five types of traditional Chinese medicine （TCM） syndrome. MethodsA total of 144 patients with HBV-ACLF were included and divided into five types of TCM syndrome， including 34 cases of heat-toxin amassment syndrome， 44 cases of dampness-heat amassment syndrome， 27 cases of Qi-deficiency and stasis jaundice syndrome， 21 cases of spleen-kidney Yang deficiency syndrome， and 18 cases of liver-kidney Yin deficiency syndrome. Meanwhile， 30 healthy volunteers were included as controls. The frequency of Treg and Th17 cells in the peripheral blood of subjects in each group was detected by flow cytometry， and the Treg/Th17 ratio was calculated. Cytometric bead array （CBA） was used to detect the levels of cytokines interleukin （IL）-10， transforming growth factor-β （TGF-β）， tumor necrosis factor-α （TNF-α）， IL-17A， and IL-23. Real-time fluorescence quantitative PCR （Real-time PCR） detected the mRNA expression of forkhead box P3 （FoxP3） and retinoic acid-related orphan receptor gamma t （ROR-γt）. Results（1） Compared with that in the healthy control group， the frequency of Treg and Th17 cells in patients with various TCM syndrome types of HBV-ACLF increased （P<0.05）. Compared with that in the heat-toxin amassment syndrome group， the frequency of Treg and Th17 cells decreased in the dampness-heat amassment syndrome group （P<0.05）， while the frequency of Treg and Th17 cells increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the dampness-heat amassment syndrome group， the frequency of Treg and Th17 cells increased in the dampness-heat amassment syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the Qi-deficiency and stasis jaundice syndrome group， the frequency of Treg and Th17 cells increased in the spleen-kidney Yang deficiency syndrome group （P<0.05）， while the frequency of Treg and Th17 cells decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with the spleen-kidney Yang deficiency syndrome group， the frequency of Treg and Th17 cells decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. （2） Compared with that in the healthy control group， the Treg/Th17 cell ratio in patients with various TCM syndromes of HBV-ACLF decreased （P<0.05）. Compared with that in the heat-toxin amassment syndrome group， the Treg/Th17 cell ratio increased in the dampness-heat amassment syndrome group （P<0.05）， while it decreased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the dampness-heat amassment syndrome group， the Treg/Th17 cell ratio decreased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the Qi-deficiency and stasis jaundice syndrome group， the Treg/Th17 cell ratio increased in the spleen-kidney Yang deficiency syndrome group and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with the spleen-kidney Yang deficiency syndrome group， the Treg/Th17 cell ratio in the liver-kidney Yin deficiency syndrome group increased （P<0.05）. （3） Compared with those in the healthy control group， the levels of Treg-related cytokines IL-10 and TGF-β， as well as Th17-related cytokines TNF-α， IL-17A， and IL-23， were elevated in patients with various TCM syndrome types of HBV-ACLF （P<0.05）. There was no significant difference in TNF-α levels among different TCM syndrome types. Compared with those in the heat-toxin amassment syndrome group， the levels of IL-10， TNF-β， IL-17A， and IL-23 in the dampness-heat amassment syndrome group， Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome groups increased （P<0.05）. Compared with those in the dampness-heat amassment syndrome group， the levels of IL-10， TGF-β， IL-17A， and IL-23 increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with those in the Qi-deficiency and stasis jaundice syndrome group， the levels of IL-10， TGF-β， IL-17A， and IL-23 in the spleen-kidney Yang deficiency syndrome group increased （P<0.05）， while those in the liver-kidney Yin deficiency syndrome group decreased （P<0.05）. Compared with those in the spleen-kidney Yang deficiency syndrome， the levels of IL-10， TGF-β， IL-17A， and IL-23 in the liver-kidney Yin deficiency syndrome group decreased （P<0.05）. （4） Compared with that in the healthy control group， the mRNA of Treg/Th17 cell specific transcription factors FoxP3 and ROR-γt were elevated in patients with various TCM syndrome types of HBV-ACLF （P<0.05）. Compared with that in the heat-toxin amassment syndrome group， the mRNA of FoxP3 and ROR-γt increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the dampness-heat amassment syndrome group， the mRNA of FoxP3 and ROR-γt increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome （P<0.05）. Compared with that in the Qi-deficiency and stasis jaundice syndrome group， the mRNA of FoxP3 and ROR-γt in the spleen-kidney Yang deficiency syndrome group increased （P<0.05）， and it decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the spleen-kidney Yang deficiency syndrome group， the mRNA of FoxP3 and ROR-γt decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. ConclusionThe frequency and ratio of Treg/Th17 cells， as well as the expression of related cytokines and specific receptors in peripheral blood of patients with HBV-ACLF in five types of TCM syndromes are different， which has certain reference value for TCM syndrome differentiation and treatment of patients with HBV-ACLF.