Qizhu anticancer prescription enhances immunosurveillance of liver cancer cells by regulating p21-dependent secretory phenotypes.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, largely due to the limitations of available therapeutic strategies. The traditional Chinese medicine Qizhu Anticancer Prescription (QZACP) can improve the quality of life and prolong the survival time of patients with HCC. However, the precise mechanisms underlying the anti-cancer properties of QZACP remain unclear. PURPOSE: This study examined the anti-hepatocarcinogenic properties of QZACP, with a specific focus on its influence on the p21-activated secretory phenotype (PASP)-mediated immune surveillance, to elucidate the underlying molecular pathways involved in HCC. MATERIALS AND METHODS: Cell proliferation was measured using the Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, and clonogenic assays. The cell cycle was evaluated using flow cytometry, and senescence was identified by staining with senescence-associated beta-galactosidase (SA-ß-gal). A primary liver cancer model produced by diethylnitrosamine was established in C57 BL/6 mice to assess the tumor-inhibitory effect of QZACP. The liver's pathological characteristics were examined using hematoxylin and eosin staining. PASP screening was performed using GeneCards, DisGeNet, Online Mendelian Inheritance in Man, and The Cancer Genome Atlas databases. Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and Transwell migration assays were performed. RESULTS: Serum containing QZACP enhanced p21 expression, triggered cell cycle arrest, accelerated cell senescence, and suppressed cell proliferation in Huh7 and MHCC-97H liver cancer cells. QZACP reduced the quantity and dimensions of liver tumor nodules and enhanced p21 protein expression, SA-ß-Gal staining in tumor lesions, and cytotoxic CD8+ T cell infiltration. Bioinformatic analyses indicated that PASP factors, including hepatocyte growth factor, decorin (DCN), dermatopontin, C-X-C motif chemokine ligand 14 (CXCL14), and Wnt family member 2 (WNT2), play an important role in the development of HCC. In addition, these factors are associated with the presence of natural killer cells and CD8+ T cells within tumors. Western blotting and ELISA confirmed that QZACP increased DCN, CXCL14, and WNT2 levels in tumor tissues and peripheral blood. CONCLUSIONS: QZACP's suppression of HCC progression may involve cell senescence mediated via p21 upregulation, DCN, CXCL14, and WNT2 secretion, and reversal of the immunosuppressive microenvironment. This study provides insights that can be used in the development of new treatment strategies for HCC.

Exosomal ncRNAs: Multifunctional contributors to the immunosuppressive tumor microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignant liver cancer characterized by aggressive progression, unfavorable prognosis, and an increasing global health burden. Therapies that precisely target immunological checkpoints and immune cells have gained significant attention as possible therapeutics in recent years. In truth, the efficacy of immunotherapy is heavily contingent upon the tumor microenvironment (TME). Recent studies have indicated that exosomes serve as a sophisticated means of communication among biomolecules, executing an essential part in the TME of immune suppression. Exosomal non-coding RNAs (ncRNAs) can induce the activation of tumor cells and immunosuppressive immune cells that suppress the immune system, such as cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), CD+8 T cells, regulatory T cells (Tregs), and regulatory B cells (Bregs). This cell-cell crosstalk triggered by exosomal ncRNAs promotes tumor proliferation and metastasis, angiogenesis, malignant phenotype transformation, and drug resistance. Hence, it is imperative to comprehend how exosomal ncRNAs regulate tumor cells or immune cells within the TME to devise more comprehensive and productive immunotherapy programs. This study discusses the features of exosomal ncRNAs in HCC and how the activation of the exosomes redefines the tumor's immunosuppressive microenvironment, hence facilitating the advancement of HCC. Furthermore, we also explored the potential of exosomal ncRNAs as a viable biological target or natural vehicle for HCC therapy.

Investigating the molecular mechanism of Qizhu anticancer prescription in inhibiting hepatocellular carcinoma based on high-resolution mass spectrometry and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Of all primary liver cancer cases, hepatocellular carcinoma (HCC) accounts for about 90%. Most patients with HCC receive a diagnosis in the medium-to-late stages or with chronic liver disease, have lost the opportunity for radical treatment, such as surgical resection, and their 5-year survival rate is low. Qizhu Anticancer Prescription (QZACP) is an empirical formula composed of traditional Chinese herbs that can clinically relieve HCC symptoms, inhibit the progression of HCC, reduce recurrence rate, and prolong survival; however, its exact mode of action remains unknown. AIM OF THE STUDY: This study's purpose was to investigate the mode of action of QZACP in the prevention and treatment of HCC. MATERIALS AND METHODS: Initially, drug components in the QZACP decoction were analyzed using high-resolution mass spectrometry. A subcutaneous tumor xenograft model in nude mice was constructed to further analyze the active components of QZACP that had entered tumor tissues through oral administration. Potential targets of QZACP in the prevention and treatment of HCC were identified and then confirmed in vivo via network pharmacology and molecular docking. In addition, regulatory effects of QZACP on HCC cell proliferation and the cell cycle were detected using a CCK-8 assay and flow cytometry. RESULTS: High-resolution mass spectrometry revealed that the QZACP decoction contained deacetyl asperulosidic acid methyl ester (DAAME), paeoniflorin, calycosin-7-glucoside, liquiritin, glycyrrhizic acid, astragaloside IV, saikosaponin A, curdione, and atractylenolide II. In nude mice, QZACP could effectively inhibit the growth of subcutaneous tumors, where DAAME, paeoniflorin, liquiritin, and glycyrrhizic acid could enter liver cancer tissues after oral administration. Among these, DAAME was the most highly expressed in HCC tissues and may be an important active component of QZACP for inhibiting HCC. Utilizing network pharmacology, the targets of action of these four drug components were identified. After verification using western blotting, STAT3, VEGFA, JUN, FGF2, BCL2L1, AR, TERT, MMP7, MMP1, ABCB1, CA9, and ESR2 were identified as targets of QZACP inhibition in HCC. In vitro experiments revealed that QZACP inhibited the proliferation of HCC cells while inducing G0/G1 phase cell cycle arrest. In vivo experiments demonstrated that DAAME significantly inhibited HCC growth. After intersection of the 24 DAAME targets predicted using network pharmacology with the 435 HCC disease targets, only CA9 was identified as a DAAME-HCC crossover target. Molecular docking results revealed that the binding site of DAAME and CA9 had good stereo-complementarity with a docking score of -8.1 kcal/mol. Western blotting and immunohistochemical results also confirmed that DAAME significantly decreased CA9 protein expression in HCC. CONCLUSIONS: QZACP inhibits HCC by reducing the expression of STAT3, VEGFA, JUN, FGF2, BCL2L1, AR, TERT, MMP7, MMP1, ABCB1, CA9, and ESR2. DAAME may be an important active component of QZACP for the prevention and treatment of HCC, inhibiting it by targeting the expression of CA9.

State of CD8+ T cells in progression from nonalcoholic steatohepatitis to hepatocellular carcinoma: From pathogenesis to immunotherapy.

With the obesity epidemic, nonalcoholic steatohepatitis (NASH) is emerging as the fastest growing potential cause of hepatocellular carcinoma (HCC). NASH has been demonstrated to establish a tumor-prone liver microenvironment where both innate and adaptive immune systems are involved. As the most typical anti-tumor effector, the cell function of CD8+ T cells is remodeled by chronic inflammation, metabolic alteration, lipid toxicity and oxidative stress in the liver microenvironment along the NASH to HCC transition. Unexpectedly, NASH may blunt the effect of immune checkpoint inhibitor therapy against HCC due to the dysregulated CD8+ T cells. Growing evidence has supported that NASH is likely to facilitate the state transition of CD8+ T cells with changes in cell motility, effector function, metabolic reprogramming and gene transcription according to single-cell sequencing. However, the mechanistic insight of CD8+ T cell states in the NASH-driven HCC is not comprehensive. Herein, we focus on the characterization of state phenotypes of CD8+ T cells with both functional and metabolic signatures in NASH-driven fibrosis and HCC. The NASH-specific CD8+ T cells are speculated to mainly have a dualist effect, where its aberrant activated phenotype sustains chronic inflammation in NASH but subsequently triggers its exhaustion in HCC. As the exploration of CD8+ T cells on the distribution and phenotypic shifts will provide a new direction for the intervention strategies against HCC, we also discuss the implications for targeting different phenotypes of CD8+ T cells, shedding light on the personalized immunotherapy for NASH-driven HCC.

Sogatella furcifera Saliva Mucin-like Protein Is Required for Feeding and Induces Rice Defences.

The white-backed planthopper (WBPH), Sogatella furcifera, is one of the most important piercing-sucking pests of rice (Oryza sativa) in Asia. Mucin-like salivary protein (SFMLP) is highly expressed in the salivary glands of WBPH, which plays an important role in WBPH feeding. In this study, WBPH injected with dsSFMLP had difficulty in sucking phloem sap from rice plants, which significantly reduced their food intake, weight, and survival. In contrast, the knockdown of the SFMLP gene had only a marginal effect on the survival of WBPH fed an artificial diet. Further studies showed that silencing SFMLP resulted in the short and single-branched salivary sheaths secretion and less formation of salivary flanges in rice. These data suggest that SFMLP is involved in the formation of the salivary sheath and is essential for feeding in WBPH. Overexpression of the SFMLP gene in rice plants promoted the feeding of WBPH, whereas silencing the gene in rice plants significantly decreased WBPH performance. Additionally, it was found that overexpression of SFMLP in rice plants elicited the signalling pathway of SA (salicylic acid) while suppressing JA (jasmonic acid); in contrast, silencing of the SFMLP gene in rice plants showed the opposite results. This study clarified the function of SFMLP in WBPH feeding as well as mediating rice defences.

Expression Analyses of Vitellogenin and Target of Rapamycin of Sogatella furcifera (Hemiptera: Delphacidae), and Their Effects on Reproduction.

Vitellogenin (Vg) and the target of rapamycin (TOR) are important genes involved in insect reproduction regulation. In this work, the full lengths of the Vg (SfVg) and TOR (SfTOR) genes of the white-backed planthopper Sogatella furcifera were cloned. The expression pattern in females showed that SfVg was highly expressed in fat bodies, and SfTOR was highly expressed in Malpighian tubules. After silencing SfVg or SfTOR, female adults did not deposit eggs. Their ovarian development was delayed, and yolk protein deposition in the oocytes was reduced. However, wild-type females mated with SfTOR-silenced males could lay eggs and produce offsprings normally. The dissections of testes and accessory glands of males with SfTOR knockdown showed that their development was not affected. Therefore, the silencing of the SfVg or SfTOR genes can effectively inhibit female reproduction, but SfTOR knockdown has no significant effect on male reproductive capacity. Furthermore, silencing SfTOR can cause SfVg expression to decrease significantly. All of the above results revealed that SfVg and SfTOR are essential for white-backed planthopper reproduction and may provide a potential target for pest control.

Chronic atrophic gastritis and intestinal metaplasia induced by high-salt and N-methyl-N'-nitro-N-nitrosoguanidine intake in rats.

The aim of the present study was to induce chronic atrophic gastritis (CAG) with intestinal metaplasia (IM) in rats by administering saturated salt and methyl-N'-nitro-N-nitrosoguanidine (MNNG) via oral gavage. Changes in gastric mucosal blood microcirculation and activation of the cyclo-oxygenase-2 (COX-2)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway during CAG and IM development were investigated. After administering saturated salt and MNNG for 25 weeks, mild atrophy was detected in the stomach of model rats using hematoxylin and eosin staining. CAG with IM was successfully induced in the gastric mucosa of the model rats after 35 weeks. Gastric mucosal blood flow was decreased in comparison with controls as early as 15 weeks after treatment to induce CAG and the mRNA expression levels of COX-2, HIF-1α, vascular endothelial growth factor receptor (VEGFR)1 and VEGFR2 were increased in comparison with untreated rats as early as 25 weeks after treatment. HIF-1α, COX-2 and VEGFR2 expression levels were increased as early as 25 weeks after CAG induction treatment when compared to controls and HIF-1α, COX-2, VEGFR1 and VEGFR2 expression levels were significantly increased after 35 weeks. These findings indicated that administering saturated salt and MNNG by gavage for 35 weeks successfully induced CAG and IM in rats. Furthermore, the microcirculation was disturbed before activation of the COX-2/HIF-1α/VEGF signaling pathway.

Weiqi Decoction Attenuated Chronic Atrophic Gastritis with Precancerous Lesion through Regulating Microcirculation Disturbance and HIF-1α Signaling Pathway.

AIM: Chronic atrophic gastritis (CAG), the precancerous lesions of gastric cancer, plays an important role in the stepwise process of gastric cancer. The ancient Chinese medicine believes in that Qi deficiency and blood stasis are involved in the pathogenesis of CAG. Weiqi decoction, a classical formula from Longhua Hospital, could supplement Qi and activate blood circulation of human beings and has been used for treating CAG in clinic over twenty years. The study aims to clarify the effect and underlying molecular mechanism of Weiqi decoction on CAG rats. METHODS: Forty-eight male Wistar rats were divided randomly into six groups: control group, model group, folic acid group, and WQD-treated groups at doses of 4 g/kg, 2 g/kg, and 1 g/kg, with eight rats in each group. MNNG and saturated NaCl were used to induce CAG rat with precancerous lesion (intestinal metaplasia and dysplasia). After 40 weeks, gastric mucosal blood flow was measured using Laser Doppler Flowmetry. The pathological changes of the gastric mucosa were identified by H&E staining and AB-PAS staining. The protein expression of COX-2, HIF-1α, VEGFR1, VEGFR2, Ki67, and cleaved caspase 3 in the gastric tissues was measured by western blotting approach. Gene expression of COX-2, HIF-1α, VEGF, VEGFR1, VEGFR2, Ang-1, and Ang-2 was detected by using Quantitative PCR method. The PGE2 concentrations in serum were detected by ELISA method. The protein expression of Ki67 in gastric mucosa was also detected by immunohistochemistry. RESULTS: Compared with control rats, atrophy and intestinal metaplasia as well as the microcirculation disturbance of gastric mucosa were induced in the stomach of CAG rats identified by the H&E and AB-PAS staining as well as microcirculation measurement, which could be significantly attenuated by WQD treatment. Moreover, compared with the control group, the protein and gene expression of COX-2, HIF-1α, VEGFR1, and VEGFR2 in gastric tissues of pylorus was obviously increased and the serum PGE2 level was significantly deceased in CAG rats, which could be significantly counteracted by WQD administration. However, the gene expression of Ang-1 and Ang-2 was not significant difference between control rats and CAG rats, and WQD also had no significant effect on the gene expression of Ang-1 and Ang-2. Furthermore, the increased cell proliferation marked by upregulated protein expression of Ki67 and decreased cell apoptosis marked by downregulated protein expression of cleaved caspase 3 in stomach of pylorus in CAG rats were obviously reversed by WQD treatment. CONCLUSION: WQD attenuated CAG with precancerous lesion through regulating gastric mucosal blood flow disturbance and HIF-1α signaling pathway.