Valproic Acid Thermally Destabilizes and Inhibits SpyCas9 Activity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system plays an important role in prokaryotic adaptive immunity. Due to its capacity for sequence-specific gene editing, CRISPR-Cas9 has become one of the most important tools widely used for genome editing in molecular biotechnology. However, its clinical application is currently limited by unwanted mutations at off-target sites. Various strategies have been developed for precise control of Cas9 in order to reduce these off-target effects, including chemical-based approaches. From a chemical screening, I observed that valproic acid (VPA) binds to and destabilizes Streptococcus pyogenes Cas9 (SpyCas9) protein in vitro, as well as in cells, while within its therapeutical concentration range under conditions of hyperthermia as demonstrated. Conditions were generated either by an external heat bag or in combination with the photothermal therapeutic agent indocyanine green activated by a near-infrared laser. Use of other histone deacetylase inhibitors failed, suggesting a histone deacetylase inhibition-independent function of VPA. Thus, this finding provides an uncomplicated thermotherapeutical approach for timely regulation of the activity of the CRISPR-Cas9 system at desired locations.

Lethal and sublethal effect of heat shock on Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

Temperature is an important abiotic environmental factor, and is responsible for various kinds of behavioral and physiological changes in living organisms. Induced heat shock is associated with feeding behaviour, reproduction and reactive oxygen species (ROS) generation that causes oxidative damage. In this experiment, we examined the lethal and sublethal effects of heat shock on reproduction, feeding behaviour and antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD) and peroxidases (POD) in P. solenopsis. Results showed that males were highly susceptible to heat shock treatments than females, as LTemp50 values were 43.8 °C for males and 45.11 °C for females. Heat shock events non-significantly affected the fecundity in female only treated adults and significantly affected the both sexes heat treated adults, it increased the xylem feeding duration, percentage of xylem feeding adults and reduce the phloem feeding duration and percentage of phloem feeding adults. Similarly it alter the antioxidant enzymes activities, an increase of CAT, SOD and POD activities were noticed in response to highest intensity of heat shock while a reduction of CAT and SOD activity were noticed in response to lowest intensity of heat shock compared to control (30 °C). These results suggest that heat shock may result in loss of body water and induce oxidative stress in P. solenopsis. However, antioxidant enzymes play a significant role in overcoming the oxidative damage.

Acidic stress induced G1 cell cycle arrest and intrinsic apoptotic pathway in Jurkat T-lymphocytes.

BACKGROUND: Low extracellular pH (pHe) is a common hallmark of tumor microenvironment, which will also affect pH sensitive T-lymphocytes in this environment. Due to the growing interest on T-cell mediated cancer therapies, acidic stress induced consequences on this lymphocyte deserves through investigations. RESULTS: In line with our previous study [Kim et al., Biochem. Biophys. Res. Commun. 2016; 472(4): 585-91.], we applied sub-lethal acidic stress (pH 3.3, 37°C for 25min) to Jurkat T-lymphocytes. Progression from early apoptosis into late apoptosis was clearly observed by flow cytometry within 3 days. Treatment led to onset of G1 arrest in the first 24h and cell cycling data corresponded to survival of an invasive alkaline phosphatase (AP) positive population. Concerning the massive cell death observed after 72h, both mRNA level (qRT-PCR) and protein level (western blotting) data indicate programmed cell death through p53-p21 independent signaling. CONCLUSION: Taken together, the results obtained suggest that the majority of Jurkat cells exposed to short but intense acidic stress conditions, as used here, undergo intrinsic apoptosis, while invasion and AP activation only occurred in a small surviving cell population.

Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation.

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

A Ruthenium(II) N-Heterocyclic Carbene (NHC) Complex with Naphthalimide Ligand Triggers Apoptosis in Colorectal Cancer Cells via Activating the ROS-p38 MAPK Pathway.

The p38 MAPK pathway is known to influence the anti-tumor effects of several chemotherapeutics, including that of organometallic drugs. Previous studies have demonstrated the important role of p38 both as a regulator and a sensor of cellular reactive oxygen species (ROS) levels. Investigating the anti-cancer properties of novel 1,8-naphthalimide derivatives containing Rh(I) and Ru(II) N-heterocyclic carbene (NHC) ligands, we observed a profound induction of ROS by the complexes, which is most likely generated from mitochondria (mtROS). Further analyses revealed a rapid and consistent activation of p38 signaling by the naphthalimide-NHC conjugates, with the Ru(II) analogue-termed MC6-showing the strongest effect. In view of this, genetic as well as pharmacological inhibition of p38α, attenuated the anti-proliferative and pro-apoptotic effects of MC6 in HCT116 colon cancer cells, highlighting the involvement of this signaling molecule in the compound's toxicity. Furthermore, the influence of MC6 on p38 signaling appeared to be dependent on ROS levels as treatment with general- and mitochondria-targeted anti-oxidants abrogated p38 activation in response to MC6 as well as the molecule's cytotoxic- and apoptogenic response in HCT116 cells. Altogether, our results provide new insight into the molecular mechanisms of naphthalimide-metal NHC analogues via the ROS-induced activation of p38 MAPK, which may have therapeutic interest for the treatment of various cancer types.

Methylisoindigo and Its Bromo-Derivatives Are Selective Tyrosine Kinase Inhibitors, Repressing Cellular Stat3 Activity, and Target CD133+ Cancer Stem Cells in PDAC.

Indirubin is an active component of the herbal ingredient 'Danggui Longhui wan', which was used for the treatment of inflammation and chronic myeloid leukemia in China. The recent study showed its derivative methylisoindigo (also known as meisoindigo) preferentially targeting cancer stem cells (CSCs) in interference with AMPK and LKB1, the cellular metabolic sensors. In this study, we screened the effect of meisoindigo on a panel of 300 protein kinases and found that it selectively inhibited Stat3-associated tyrosine kinases and further confirmed its activity in cell based assays. To gain a deeper insight into the structure-activity relationship we produced 7 bromo-derivatives exhausting the accessible positions on the bisindole backbone except for in the 4-position due to the space limitation. We compared their anti-proliferative effects on tumor cells. We found that 6-bromomeisoindigo showed improved toxicity in company with increased Stat3 inhibition. Moreover, we detected that 6-bromomeisoindigo induced apoptosis of 95% of CD133+ pancreatic cancer cells. Considering that CD133 is a common marker highly expressed in a range of CSCs, our results imply the potential application of 6-bromomeisoindigo for the treatment of CSCs in different types of cancers.

Modified STAP conditions facilitate bivalent fate decision between pluripotency and apoptosis in Jurkat T-lymphocytes.

Low extracellular pH (pHe) is not only the result of cancer metabolism, but a factor of anti-cancer drug efficacy and cancer immunity. In this study, the consequences of acidic stress were evaluated by applying STAP protocol on Jurkat T-lymphocytes (2.0 × 10(6) cells/ml, 25 min in 37 °C). We detected apoptotic process exclusively in pH 3.3 treated cells within 8 h with western blotting (WB). This programmed cell death led to significant drop of cell viability in 72 h measured by MTT assay resulting PI positive population on flow cytometry (FCM) at day 7. Quantified RT-PCR (qRT-PCR) data indicated that all of above mentioned responses are irrelevant to expression of OCT4 gene variants. Interestingly enough, pluripotent cells represented by positive alkaline phosphatase (AP) staining survived acidic stress and consequently proportion of AP positive cells was significantly increased after pH 3.3 treatment (day 7). In general, acidic treatment led to an apoptotic condition for Jurkat T-lymphocytes, which occurred independent of OCT4 induction.

The Role of Indirubins in Inflammation and Associated Tumorigenesis.

Indirubin is the major active component of an herbal recipe 'Dangui Luhui Wan' () in traditional Chinese medicine (TCM). It is widely used in China for the treatment of inflammation, cancer, and other chronic diseases and is known for good efficiency and very low side effects. Primary studies on the mechanism of action revealed that indirubin and derivatives are potent ATP-competitive inhibitors of CDKs and GSK3ß achieving IC50 values down to the low nanomolar range. However, the clinical application of indirubins is limited by the extremely poor water solubility (<1 mg/L in general) and consequently the insufficient bioavailability originating from strong binding forces in the crystal lattice. In the last few decades, a lot of efforts had been put into the structure optimization of indirubin derivatives binding selectively to specific kinases. Thus, a number of new indirubins have been developed bearing substituents mainly in the 5- and 3'-position suitable for improved solubility and inhibition against CDKs and GSK3ß, referred to as canonical indirubins. Interestingly, several noncanonical 7- and 7'-indirubin derivatives have been reported, showing a distinct binding model in the ATP-binding pocket and targeting a very different spectrum of protein kinases as seen from kinase profiling. In this chapter, we will review the field of indirubin research from its discovery, synthesis, chemical modification, structure-activity relationship, and mechanism of action to molecular targets comprising recent advantages and new findings in the context of inflammation-associated signaling pathways, in particular in tumorigenesis, including NF-κB, STAT3, TGF-ß, and AhR.

Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.

Bone morphogenetic proteins (BMPs) are members of the TGFß family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFß receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and time-dependent manner, which can provide an explanation of some properties of the BMP regulatory network.

A TrxR inhibiting gold(I) NHC complex induces apoptosis through ASK1-p38-MAPK signaling in pancreatic cancer cells.

BACKGROUND: Cancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive ROS resulting in the compensation for intrinsic oxidative stress and therapeutic resistance. PDAC is one of the most lethal cancers and often associated with a high accumulation of ROS. Recent studies identified gold(I) NHC complexes as potent TrxR inhibitors suppressing cell growth in a wide spectrum of human malignant cell lines at the low micromolar concentration. However, the mechanism of action is not completely elucidated yet. METHODS: To understand the biological function of gold(I) NHC complexes in PDAC, we used a recently published gold(I) NHC complex, MC3, and evaluated its anti-proliferative effect in four PDAC cell lines, determined by MTT and SRB assays. In further detailed analysis, we analyzed cellular ROS levels using the ROS indicator DHE and mitochondrial membrane potential indicated by the dye JC-1 in Panc1. We also analyzed cell cycle arrest and apoptosis by FACS. To elucidate the role of specific cell signaling pathways in MC3-induced cell death, co-incubation with ROS scavengers, a p38-MAPK inhibitor and siRNA mediated depletion of ASK1 were performed, and results were analyzed by immunoblotting, ELISA-microarrays, qRT-PCR and immunoprecipitation. RESULTS: Our data demonstrate that MC3 efficiently suppressed cell growth, and induced cell cycle arrest and apoptosis in pancreatic cancer cells, in particular in the gemcitabine-resistant cancer cells Panc1 and ASPC1. Treatment with MC3 resulted in a substantial alteration of the cellular redox homeostasis leading to increased ROS levels and a decrease in the mitochondrial membrane potential. ROS scavengers suppressed ROS formation and rescued cells from damage. On the molecular level, MC3 blocked the interaction of Trx with ASK1 and subsequently activated p38-associated signaling. Furthermore, inhibition of this pathway by using ASK1 siRNA or a p38 inhibitor clearly attenuated the effect of MC3 on cell proliferation in Panc1 and ASPC1. CONCLUSIONS: Our results confirm that MC3 is a TrxR inhibitor and show MC3 induced apoptosis in gemcitabine-resistant PDACs. MC3 mediated cell death could be blocked by using anti-oxidants, ASK1 siRNA or p38 inhibitor suggesting that the Trx-ASK1-p38 signal cascade played an important role in gold(I) NHC complexes-mediated cellular damage.

7,7'-Diazaindirubin--a small molecule inhibitor of casein kinase 2 in vitro and in cells.

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7'-diazaindigo (10) and 7,7'-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 µM, 7-azaindirubin (12) and 7'-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7'-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 µM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.

Recent Advances in Covalent Drug Discovery.

In spite of the increasing number of biologics license applications, the development of covalent inhibitors is still a growing field within drug discovery. The successful approval of some covalent protein kinase inhibitors, such as ibrutinib (BTK covalent inhibitor) and dacomitinib (EGFR covalent inhibitor), and the very recent discovery of covalent inhibitors for viral proteases, such as boceprevir, narlaprevir, and nirmatrelvir, represent a new milestone in covalent drug development. Generally, the formation of covalent bonds that target proteins can offer drugs diverse advantages in terms of target selectivity, drug resistance, and administration concentration. The most important factor for covalent inhibitors is the electrophile (warhead), which dictates selectivity, reactivity, and the type of protein binding (i.e., reversible or irreversible) and can be modified/optimized through rational designs. Furthermore, covalent inhibitors are becoming more and more common in proteolysis, targeting chimeras (PROTACs) for degrading proteins, including those that are currently considered to be 'undruggable'. The aim of this review is to highlight the current state of covalent inhibitor development, including a short historical overview and some examples of applications of PROTAC technologies and treatment of the SARS-CoV-2 virus.

Molecular Marvels: Small Molecules Paving the Way for Enhanced Gene Therapy.

In the rapidly evolving landscape of genetic engineering, the advent of CRISPR-Cas technologies has catalyzed a paradigm shift, empowering scientists to manipulate the genetic code with unprecedented accuracy and efficiency. Despite the remarkable capabilities inherent to CRISPR-Cas systems, recent advancements have witnessed the integration of small molecules to augment their functionality, introducing new dimensions to the precision and versatility of gene editing applications. This review delves into the synergy between CRISPR-Cas technologies based specifically on Cas9 and small-molecule drugs, elucidating the pivotal role of chemicals in optimizing target specificity and editing efficiency. By examining a diverse array of applications, ranging from therapeutic interventions to agricultural advancements, we explore how the judicious use of chemicals enhances the precision of CRISPR-Cas9-mediated genetic modifications. In this review, we emphasize the significance of small-molecule drugs in fine-tuning the CRISPR-Cas9 machinery, which allows researchers to exert meticulous control over the editing process. We delve into the mechanisms through which these chemicals bolster target specificity, mitigate off-target effects, and contribute to the overall refinement of gene editing outcomes. Additionally, we discuss the potential of chemical integration in expanding the scope of CRISPR-Cas9 technologies, enabling tailored solutions for diverse genetic manipulation challenges. As CRISPR-Cas9 technologies continue to evolve, the integration of small-molecule drugs emerges as a crucial avenue for advancing the precision and applicability of gene editing techniques. This review not only synthesizes current knowledge but also highlights future prospects, paving the way for a deeper understanding of the synergistic interplay between CRISPR-Cas9 systems and chemical modulators in the pursuit of more controlled and efficient genetic modifications.

Development of a next-generation endogenous OCT4 inducer and its anti-aging effect in vivo.

The identification of small molecules capable of replacing transcription factors has been a longstanding challenge in the generation of human chemically induced pluripotent stem cells (iPSCs). Recent studies have shown that ectopic expression of OCT4, one of the master pluripotency regulators, compromised the developmental potential of resulting iPSCs, This highlights the importance of finding endogenous OCT4 inducers for the generation of clinical-grade human iPSCs. Through a cell-based high throughput screen, we have discovered several new OCT4-inducing compounds (O4Is). In this work, we prepared metabolically stable analogues, including O4I4, which activate endogenous OCT4 and associated signaling pathways in various cell lines. By combining these with a transcription factor cocktail consisting of SOX2, KLF4, MYC, and LIN28 (referred to as "CSKML") we achieved to reprogram human fibroblasts into a stable and authentic pluripotent state without the need for exogenous OCT4. In Caenorhabditis elegans and Drosophila, O4I4 extends lifespan, suggesting the potential application of OCT4-inducing compounds in regenerative medicine and rejuvenation therapy.

pVHL-mediated SMAD3 degradation suppresses TGF-ß signaling.

Transforming growth factor ß (TGF-ß) signaling plays a fundamental role in metazoan development and tissue homeostasis. However, the molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-ß signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, we identify pVHL as an E3 ligase for SMAD3 ubiquitination. We show that pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-ß targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-ß/SMAD signaling. Thus, our study demonstrates a conserved role of pVHL in the regulation of TGF-ß/SMAD3 signaling in human cells and Drosophila wing development.

A PROTAC targets splicing factor 3B1.

The proteolysis-targeting chimeras (PROTACs) are a new technology to degrade target proteins. However, their clinical application is limited currently by lack of chemical binders to target proteins. For instance, it is still unknown whether splicing factor 3B subunit 1 (SF3B1) is targetable by PROTACs. We recently identified a 2-aminothiazole derivative (herein O4I2) as a promoter in the generation of human pluripotent stem cells. In this work, proteomic analysis on the biotinylated O4I2 revealed that O4I2 targeted SF3B1 and positively regulated RNA splicing. Fusing thalidomide-the ligand of the cereblon ubiquitin ligase-to O4I2 led to a new PROTAC-O4I2, which selectively degraded SF3B1 and induced cellular apoptosis in a CRBN-dependent manner. In a Drosophila intestinal tumor model, PROTAC-O4I2 increased survival by interference with the maintenance and proliferation of stem cell. Thus, our finding demonstrates that SF3B1 is PROTACable by utilizing noninhibitory chemicals, which expands the list of PROTAC target proteins.

A Chemical Toolbox for Labeling and Degrading Engineered Cas Proteins.

The discovery of clustered regularly interspaced short palindromic repeats and their associated proteins (Cas) has revolutionized the field of genome and epigenome editing. A number of new methods have been developed to precisely control the function and activity of Cas proteins, including fusion proteins and small-molecule modulators. Proteolysis-targeting chimeras (PROTACs) represent a new concept using the ubiquitin-proteasome system to degrade a protein of interest, highlighting the significance of chemically induced protein-E3 ligase interaction in drug discovery. Here, we engineered Cas proteins (Cas9, dCas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (known as the π-clamp system) and demonstrate that the modified CasFCPF proteins can be (1) labeled in live cells by perfluoroaromatics carrying the fluorescein or (2) degraded by a perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). A proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed a high target specificity, suggesting a wide range of applications of perfluoroaromatics-induced proximity in the regulation of stability, activity, and functionality of any FCPF-tagging protein.

Baicalein Induces Apoptosis of Pancreatic Cancer Cells by Regulating the Expression of miR-139-3p and miR-196b-5p.

Pancreatic cancer is a common malignant tumor with a high incidence and mortality rate. The prognosis of patients with pancreatic cancer is considerably poor due to the lack of effective treatment in clinically. Despite numerous studies have revealed that baicalein, a natural product, is responsible for suppressing multiple cancer cells proliferation, motility and invasion. The mechanism by which baicalein restraining pancreatic cancer progression remains unclear. In this study, we firstly verified that baicalein plays a critical role in inhibiting pancreatic tumorigenesis in vitro and in vivo. Then we analyzed the alteration of microRNAs (miRNAs) expression levels in Panc-1 cells incubated with DMSO, 50 and 100 µM baicalein by High-Throughput sequencing. Intriguingly, we observed that 20 and 39 miRNAs were accordingly up- and down-regulated through comparing Panc-1 cells exposed to 100 µM baicalein with the control group. Quantitative PCR analysis confirmed that miR-139-3p was the most up-regulated miRNA after baicalein treatment, while miR-196b-5p was the most down-regulated miRNA. Further studies showed that miR-139-3p induced, miR-196b-5p inhibited the apoptosis of Panc-1 cells via targeting NOB1 and ING5 respectively. In conclusion, we demonstrated that baicalein is a potent inhibitor against pancreatic cancer by modulating the expression of miR-139-3p or miR-196b-5p.

Synthesis and cytotoxicity of novel indirubin-5-carboxamides.

Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility. We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substituents with basic centers. Quaternization or protonation of these alkylamino substituents provided indirubins with significantly improved solubility without loss of bioactivity.

Yin Zhi Huang, a traditional Chinese herbal formula, ameliorates diet-induced obesity and hepatic steatosis by activating the AMPK/SREBP-1 and the AMPK/ACC/CPT1A pathways.

BACKGROUND: Yin Zhi Huang (YZH) is a formula composed of Artemisia scoparia, Gardeniae fructus, Scutellaria baicalensis Georgi, and Lonicerae Japonicae Flos. Most of the components are eaten as food in Asia. Here, we evaluated the role of YZH on a high-fat diet (HFD)-induced obesity and hepatic steatosis. METHODS: Male C57BL/6J mice were fed with normal-chow diet, HFD, and HFD with low- or high-dose YZH for 16 weeks. Body weight gain, adipose mass, and plasma lipids levels were measured to evaluate the effect of YZH on obesity. Liver weight and staining methods on liver tissues were used to determine hepatic steatosis. The expression of involved genes and proteins were screened with qRT-PCR and immunoblotting, respectively. RESULTS: The results showed that YZH significantly reduced body weight gain, adipose mass, and the size of adipocytes, while did not affect food intake in HFD-fed mice. H&E staining, bodipy staining, and oil red O staining displayed that YZH alleviates hepatic lipid accumulation. It also effectively restored elevated plasma levels of triglycerides (TG), total cholesterol (TC), alanine aminotransferase, and aspartate aminotransferase in HFD-fed mice. Mechanistically, these effects of YZH have associated with a decrease of AMPK/SREBP-1 pathway-mediated de novo lipogenesis and an increase of AMPK/ACC/CPT1A pathway-mediated mitochondrial fatty acid ß oxidation. CONCLUSIONS: YZH supplementation ameliorated diet-induced obesity and hepatic steatosis by decreasing AMPK/SREBP-1 pathway-mediated de novo lipogenesis and increasing AMPK/ACC/CPT1A pathway-mediated mitochondrial fatty acid ß oxidation.