Si-Ni-San reduces lipid droplet deposition associated with decreased YAP1 in metabolic dysfunction-associated fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most common chronic liver disease worldwide. However, its complex pathogenesis and lack of effective drugs for treating it present significant challenges. Si-Ni-San (SNS) is one of the representative formulas for treating patients with MAFLD in traditional Chinese medicine (TCM) clinics. According to our previous work, SNS reduces lipid droplet (LD) deposition in livers of mice with MAFLD. AIM OF THE STUDY: To elucidate the mechanism of SNS in reducing LD deposition in MAFLD. MATERIALS AND METHODS: First, LD areas were detected with Oil red O staining in HepG2 cells induced by oleic acid (OA). Cell Counting Kit-8 (CCK-8) assay was used to test cell viability after treatment with different concentrations of SNS serum. The expression of Yes-associated protein 1 (YAP1) was monitored by Western blot. Second, C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks and gavaged with SNS decoction during the 11th and 12th weeks. Then, the weight of the body and the liver was examined. LD numbers and their locations in the liver were detected by triglyceride (TG) assay and hematoxylin and eosin staining (H&E). The expression levels of YAP1 and perilipin2 (PLIN2) were detected using Western blot and immunohistochemistry (IHC) in liver tissues. Finally, active ingredients of SNS decoction and SNS serum were identified by liquid chromatography-mass spectrometry (LC-MS). Finally, molecular docking was performed between the compounds in SNS and YAP1 to analyze their active interaction. RESULTS: Cellular experiments showed that SNS serum reduced LD vacuoles and YAP1 expression in OA-induced HepG2 cells. Animal experiments confirmed that LD vacuoles, PLIN2 expression (3.16-fold), and YAP1 expression (2.50-fold) were increased in the HFD group compared with the normal diet (ND) group. SNS reduced LD vacuoles, TG content (0.84-fold), PLIN2 expression (0.33-fold), and YAP1 expression (0.27-fold) compared with the normal saline (NS) group in Yap1Flox mice with MAFLD. In SNS, baicalein-6-glucuronide, desoxylimonin, galangin-7-glucoside, glycyrrhizic-acid, licoricesaponin-K2, and nobiletin showed a high binding effect with YAP1. Knockout of hepatocyte YAP1 reduced LD vacuoles, TG content (0.40-fold), and PLIN2 expression (0.62-fold) in mice. Meanwhile, SNS reduced LD vacuoles, TG content (0.70-fold), and PLIN2 expression (0.19-fold) in Yap1LKO mice with MAFLD. The effect of SNS in reducing TG and PLIN2 was diminished in Yap1LKO mice compared with Yap1Flox mice. CONCLUSION: SNS reduced LD deposition and YAP1 expression in MAFLD liver cells both in vivo and in vitro. YAP1 was highly expressed in livers with MAFLD, and knockout of hepatocellular YAP1 reduced LD deposition in mice. SNS reduced LD deposition associated with decreased YAP1 in MAFLD liver cells.

Dihydroartemisinin inhibited the Warburg effect through YAP1/SLC2A1 pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) was the third most common cause of cancer death. But it has only limited therapeutic options, aggressive nature, and very low overall survival. Dihydroartemisinin (DHA), an anti-malarial drug approved by the Food and Drug Administration (FDA), inhibited cell growth in HCC. The Warburg effect was one of the ten new hallmarks of cancer. Solute carrier family 2 member 1 (SLC2A1) was a crucial carrier for glucose to enter target cells in the Warburg effect. Yes-associated transcriptional regulator 1 (YAP1), an effector molecule of the hippo pathway, played a crucial role in promoting the development of HCC. This study sought to determine the role of DHA in the SLC2A1 mediated Warburg effect in HCC. In this study, DHA inhibited the Warburg effect and SLC2A1 in HepG2215 cells and mice with liver tumors in situ. Meanwhile, DHA inhibited YAP1 expression by inhibiting YAP1 promoter binding protein GA binding protein transcription factor subunit beta 1 (GABPB1) and cAMP responsive element binding protein 1 (CREB1). Further, YAP1 knockdown/knockout reduced the Warburg effect and SLC2A1 expression by shYAP1-HepG2215 cells and Yap1LKO mice with liver tumors. Taken together, our data indicated that YAP1 knockdown/knockout reduced the SLC2A1 mediated Warburg effect by shYAP1-HepG2215 cells and Yap1LKO mice with liver tumors induced by DEN/TCPOBOP. DHA, as a potential YAP1 inhibitor, suppressed the SLC2A1 mediated Warburg effect in HCC.

Dihydroartemisinin broke the tumor immunosuppressive microenvironment by inhibiting YAP1 expression to enhance anti-PD-1 efficacy.

The efficacy of anti-PD-1 therapy is not as expected in hepatocellular carcinoma (HCC). YAP1 was overexpressed and activated in HCC. The mechanism of YAP1 in HCC immune escape is unclear. Anti-PD-1 treatment increased YAP1 expression in liver tumor cells, and exhausted CD4+ and CD8+ T cells in the blood and spleen of liver tumor mice. YAP1 knockdown suppressed PD-L1 expression, which was involved in JAK1/STAT1, 3 pathways. Moreover, Yap1 knockout elevated CD4+ and CD8+ T cells in liver tumor niche. Consistently, verteporfin, YAP1 inhibitor, decreased TGF-ß and IFN-γ in liver tumor niche and exhausted CD8+ T cell in the spleen. DHA suppressed YAP1 expression and break immune evasion in liver tumor niche, characterized by decreased PD-L1 in liver tumor cells and increased CD8+ T cell infiltration. Furthermore, DHA combined with anti-PD-1 treatment promoted CD4+ T cell infiltration in the spleen and CD8+ T cell in tumor tissues of mice. In summary, YAP1 knockdown in liver tumor cells suppressed PD-L1 expression and recruited cytotoxic T lymphocytes (CTLs), leading to break immune evasion in tumor niche. Mechanistically, YAP1 knockdown suppressed PD-L1 expression, which was involved in JAK1/STAT1, 3 pathways. Finally, DHA inhibited YAP1 expression, which not only inhibited liver tumor proliferation but also break the immunosuppressive niche in liver tumor tissues and improve the effect of anti-PD-1 therapy.

The Effects of Qinghao-Kushen and Its Active Compounds on the Biological Characteristics of Liver Cancer Cells.

Background and Aims: Artemisia annua (Qinghao) and Sophora flavescens (Kushen) are traditional Chinese medicines (TCMs). They are widely used in disease therapy, including hepatocellular carcinoma (HCC). However, their key compounds and targets for HCC treatment are unclear. This article mainly analyzed the vital active compounds and the mechanism of Qinghao-Kushen acting on HCC. Methods: First, we chose a traditional Chinese medicine, which has an excellent clinical effect on HCC by network meta-analysis. Then, we composed the Qinghao-Kushen herb pair and prepared the medicated serum. The active compounds of Qinghao-Kushen were verified by the LC-MS method. Next, we detected key targets from PubChem, SymMap, SwissTargetPrediction, DisGeNET, and GeneCards databases. Subsequently, the mechanism of Qinghao-Kushen was predicted by network pharmacology strategy and primarily examined in HuH-7 cells, HepG2 cells, and HepG2215 cells. Results: The effect of the Qinghao-Kushen combination was significantly better than that of single Qinghao or single Kushen in HepG2 and HuH-7 cells. Qinghao-Kushen increased the expression of activated caspase-3 protein than Qinghao or Kushen alone in HepG2 and HepG2215 cells. Network analyses and the LC-MS method revealed that the pivotal compounds of Qinghao-Kushen were matrine and scopoletin. GSK-3ß was one of the critical molecules related to Qinghao-Kushen. We confirmed that Qinghao-Kushen and matrine-scopoletin decreased the expression of GSK-3ß in HepG2 cells while increased GSK-3ß expression in HepG2215 cells. Conclusions: This work not only illustrated that the practical components of Qinghao-Kushen on HCC were matrine and scopoletin but shed light on the inhibitory of Qinghao-Kushen and matrine-scopoletin on liver cancer cells. Moreover, Qinghao-Kushen and matrine-scopoletin had a synergistic effect over the drug alone in HuH-7, HepG2, or HepG2215 cells. GSK-3ß may be a potential target for HCC therapy.

Modulation of Oral Microflora during Helicobacter Pylori Eradication.

OBJECTIVE: To analyse the saliva microbial abundance and composition by 16s rRNA sequence during Helicobacter pylori (H.pylori) eradication. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Hebei University of Chinese Medicine, Hebei Provincial Hospital of Traditional Chinese Medicine, from March 2019 to January 2020. METHODOLOGY: The saliva microbial were analysed before and after the bismuth-containing quadruple therapy. A total of ten saliva samples (three groups) were enrolled in the study. The authors used the linear discriminant analysis effect size (LEfSe) method and Welch's t-test for comparative analysis to identify which taxa could be significantly affected in three groups. RESULTS: H.pylori 16S rRNA gene sequence was not detected in the ten saliva samples. The abundance of Prevotella_sp._oral_clone_P4PB_83_P2 from healthy adults was higher than H.pylori-positive patients. Moreover, after the bismuth-containing quadruple therapy, the diversity and richness of saliva bacteria reduced. Lautropia, Burkholderiales, uncultured bacterium, Burkholderiaceae, and Actinomyces were enriched in H.pylori-positive patient samples after the bismuth-containing quadruple therapy. CONCLUSION: The diversity and richness of salivary microbiome were reduced in H.pylori-positive patient, and bismuth-containing quadruple therapy affected oral microbiota. Key Words: Helicobacter pylori, Saliva, Microbiota, RNA, Bismuth.

Dihydroartemisinin reduced lipid droplet deposition by YAP1 to promote the anti-PD-1 effect in hepatocellular carcinoma.

BACKGROUND: Anti-PD-1 was used to treat for many cancers, but the overall response rate of monoclonal antibodies blocking the inhibitory PD-1/PD-L1 was less than 20%. Lipid droplet (LD) deposition reduced chemotherapy efficacy, but whether LD deposition affects anti-PD-1 treatment and its mechanism remains unclear. Dihydroartemisinin (DHA) was FDA proved antimalarial medicine, but its working mechanism on LD deposition has not been clarified. PURPOSE: This study aimed to elucidate the mechanism of DHA reducing LDs deposition and improving the efficacy of anti-PD-1. METHODS: LD numbers and area were separately detected by electron microscopy and oil Red O staining. The expression of YAP1 and PLIN2 was detected by immunohistochemical staining in liver cancer tissues. Transcription and protein expression levels of YAP1 and PLIN2 in cells were detected by qRT-PCR and Western blot after DHA treated HepG2215 cells and Yap1LKO mice. RESULTS: LD accumulation was found in the liver tumor cells of DEN/TOPBCOP-induced liver tumor mice with anti-PD-1 treatment. But DHA treatment or YAP1 knockdown reduced LD deposition and PLIN2 expression in HepG2215 cells. Furthermore, DHA reduced the LD deposition, PLIN2 expression and triglycerides (TG) content in the liver tumor cells of Yap1LKO mice with liver tumor. CONCLUSION: Anti-PD-1 promoted LD deposition, while YAP1 knockdown/out reduced LD deposition in HCC. DHA reduced LD deposition by inhibiting YAP1, enhancing the effect of anti-PD-1 therapy.

Anti-malarial drug dihydroartemisinin downregulates the expression levels of CDK1 and CCNB1 in liver cancer.

Liver cancer is the third leading cause of cancer-associated mortality worldwide. By the time liver cancer is diagnosed, it is already in the advanced stage. Therefore, novel therapeutic strategies need to be identified to improve the prognosis of patients with liver cancer. In the present study, the profiles of GSE84402, GSE19665 and GSE121248 were used to screen differentially expressed genes (DEGs). Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses for DEGs were conducted using the Database for Annotation, Visualization and Integrated Discovery. The protein-protein interaction network was established to screen the hub genes associated with liver cancer. Additionally, the expression levels of hub genes were validated using the Gene Expression Profiling Interactive Analysis and Oncomine databases. In addition, the prognostic value of hub genes in patients with liver cancer was analyzed using Kaplan-Meier Plotter. It was demonstrated that 132 and 246 genes were upregulated and downregulated, respectively, in patients with liver cancer. Among these DEGs, 10 hub genes with high connected node values were identified, which were AURKA, BIRC5, BUB1B, CCNA2, CCNB1, CCNB2, CDC20, CDK1, DLGAP5 and MAD2L1. CDK1 and CCNB1 had the most connection nodes and the highest score and were therefore, the most significantly expressed. In addition, it was demonstrated that high expression levels of CDK1 and CCNB1 were associated with poor overall survival time of patients with liver cancer. Dihydroartemisinin (DHA) is a Food and Drug Administration-approved drug, which is derived from the traditional Chinese medicine Artemisia annua Linn. DHA inhibits cell proliferation in numerous cancer types, including liver cancer. In our previous study, it was revealed that DHA inhibited the proliferation of HepG2215 cells. In the present study, it was further demonstrated that DHA reduced the expression levels of CDK1 and CCNB1 in liver cancer. Overall, CDK1 and CCNB1 were the potential therapeutic targets of liver cancer, and DHA reduced the expression levels of CDK1 and CCNB1, and inhibited the proliferation of liver cancer cells.

Dihydroartemisinin, an antimalarial drug, induces absent in melanoma 2 inflammasome activation and autophagy in human hepatocellular carcinoma HepG2215 cells.

As an effective antimalarial drug, Dihydroartemisinin (DHA) is readily isolated from the traditional Chinese medicine of Artemisia annua. DHA is not only an autophagy promoter but also a substance with strong antitumor efficiency. The relationship between autophagy and inflammasomes has been suggested in hepatocellular carcinoma (HCC). However, there are few reports describing relationships between inflammasomes and autophagy in HCC therapy. The present study demonstrated that DHA suppressed cell proliferation in HepG2215 cells in a dose- and time-dependent manner. The inhibitory activity is mediated by autophagy, in which reactive oxygen species (ROS) production induced nuclear and mitochondrial DNA damage. Then, DHA were first shown to promote AIM2/caspase-1 inflammasome. Compared with the DHA group, the autophagy inhibitor 3-MA significantly inhibited the expressions of activated Caspase-1, a pyroptotic marker proteins. Meanwhile, repression of mTOR by rapamycin promoted autophagy and AIM2/caspase-1 activation. The caspase-1 inhibitor Z-YVAD-FMK also notably blocked autophagy cell death characterized by the downexpression of Beclin-1 and LC3-II. Additionally, the study demonstrated that DHA suppressed pseudopodium formation and cell mobility. Therefore, we first reveal a novel mechanism that DHA promotes AIM2/caspase-1 inflammasome, which contributes to autophagy in HepG2215 cells. Moreover, nuclear and mitochondrial DNA damage was also involved in this process via ROS production.

Antimalarial Dihydroartemisinin triggers autophagy within HeLa cells of human cervical cancer through Bcl-2 phosphorylation at Ser70.

BACKGROUND: As an effective antimalarial medicine, Dihydroartemisinin (DHA) has therapeutic potential on human cervical cancer. However, its working mechanism has not been elucidated. PURPOSE: This study aimed to investigate the reversal effect of DHA on human cervical cancer HeLa cells, and explored its mechanism of action in vitro and in vivo. STUDY DESIGN/METHODS: The effect and mechanism of DHA on HeLa cells was examined by using CCK-8 assay, flow cytometry, transmission electron microscopy, immunofluorescence, and Western blot analysis in human hepatocellular carcinoma cells. RESULTS: In this study, it was confirmed that DHA had statistically equivalent anti-tumor efficiency in HeLa cells with a clinical chemotherapeutic agent of cisplatin. Meanwhile, DHA triggered autophagy, where LC3B-II expression was dose-dependently increased. Further, it was revealed that DHA promotes reactive oxygen species (ROS) generation, with DNA double-strand breaks (DSB) damage, as up-regulation of γH2AX protein and foci formation. Interestingly, we firstly demonstrated that DHA induced autophagy through promotion of the phosphorylation of Bcl-2 (Ser70), independent of the phosphorylated JNK1/2 (Thr183/Tyr185). Moreover, DHA-treated HeLa cells displayed an increase in the pro-autophagic protein Beclin-1 with downregulated the phospho-mTOR (Ser2448). Furthermore, upregulated pro-apoptotic protein Bak-1, but not Bax, suggesting Bak-1 is included in DHA-induced autophagy. CONCLUSION: Therefore, DHA upregulates the phosphorylation of Bcl-2 (Ser70) and mTOR (Ser2448) and induces autophagic cell death in Hela cells. This study provided a mechanism to support DHA, an autophagy inducer, as a potential therapeutic agent for human cervical cancer.

Effects of magnetic dihydroartemisinin nano-liposome in inhibiting the proliferation of head and neck squamous cell carcinomas.

BACKGROUND: Dihydroartemisinin (DHA) was one of the most potent anticancer artemisinin-like compounds that had been proved by many researchers, but its application was limited by its own characteristics. PURPOSE: Magnetic DHA nano-liposomes (DHA-MLPs) were developed to improve the targeting antitumor efficiency and bioavailability of DHA, and their physical properties were characterized. STUDY DESIGN AND METHODS: Liposomes were prepared by thin film dispersion and orthogonal experimental design was used to optimize the formula. The magnetic targeting and antitumor effects of DHA-MLPs in the externally applied magnetic field was investigated in vitro and in vivo. RESULTS: The mean particle size of DHA-MLPs was 209.10 ±â€¯4.92 nm, the charge potential was -37.13 ±â€¯1.01 mV, the encapsulation efficiency (E.E.%) was 82.12 ±â€¯0.91%, and the saturation magnetization at room temperature was 11.84 emu g-1. Targeting DHA-MLPs as well as free DHA could lead to cell cycle G1 block and apoptosis of HNSCC tumor cells in vitro. The tumor volumes of targeting DHA-MLPs treated mouse group were distinctly decreased than that in the control group, free DHA group and non-targeting DHA-MLPs group (P < 0.05). It was observed from iron staining intensity that DHA-MLPs had significant targeting effect in magnetic field (P < 0.05). CONCLUSION: This novelty liposome could strengthen the ability of DHA in tumor suppression, by increasing the targeted delivery of DHA and biocompatibility, optimize the bioefficacy of DHA.

Learning from Clostridium novyi-NT: How to defeat cancer.

Side effects associated with conventional anticancer therapies have prompted the new idea of solid tumor treatment strategy. One of them is using bacteria explored as potential antitumor agents over more than one century. Notably, the ideal therapy is a specifical target to tumors with limited toxicity. Here, we take "Clostridium novyi" for the search keyword in the PubMed from 2000 to 2015 and describe that C. novyi-NT spores act as "Trojan horse" for bacteriolytic therapy. This therapy is based on the fact that the live and attenuated obligate anaerobic bacteria are capable of binary fission selectively in anoxic areas of solid tumors and direct tumoricidal effects. Our succinct review mainly concentrates on the potential mechanisms of combination bacteriolytic therapy, an effective and safe tumor therapy with the help of C. novyi-NT. Importantly, C. novyi-NT spores were shown to induce solid tumor regression and exhibit the property to initiate an immune response. Therefore, C. novyi-NT spores should be an effective and safe tumor therapy.

[The authorized considerations on the pre-clinical study of drug-eluting coronary stent system].

China Food and Drug Administration didn't issue any guideline on the pre-clinical study of drug-eluting coronary stent system, the basic requirement of the authorized administration was summarized to help manufacture prepare the document during the registration process.