In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model's efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI50 of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P < 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P < 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P < 0.05).

Exploring the inhibitory impact of Mongolian medicinal He-Zi-3 soup on mammary gland hyperplasia in rats induced by estrogen and progestogen.

ETHNOPHARMACOLOGICAL RELEVANCE: Mammary gland hyperplasia, a prevalent benign breast condition, often serves as a precursor to various other breast diseases. He-Zi-3 soup (HZ-3), a traditional Mongolian remedy, is utilized for treating this condition. AIM OF THE STUDY: To explore the effect and underlying mechanism of HZ-3, a Mongolian medicinal preparation, on mammary gland hyperplasia. MATERIALS AND METHODS: This study aimed to assess the impact of different doses of HZ-3 in a rat model of mammary hyperplasia. The active components within HZ-3 drug serum were identified and analyzed through network pharmacology and target prediction. To elucidate the underlying mechanism of HZ-3 in addressing mammary hyperplasia, we conducted a series of investigations on estradiol-induced mammary hyperplasia in model rates. Assessments included measurements of papilla width and height, hematoxylin and eosin staining, Masson staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry. RESULTS: Our investigation revealed the identification of 21 compounds, primarily terpenoids, through serum medicinal chemistry screening. Utilizing network pharmacological analysis, we observed predominant regulation through the estrogen pathway, closely associated with key genes including esr1,esr2, ncoa1, krt 19, ctsd, ebag 9, and bcl-2. Assessments encompassing nipple height and width, histological examination, immunohistochemical analysis, and serum hormone levels via enzyme-linked immunosorbent assay demonstrated the inhibitory effect of HZ-3 on mammary hyperplasia in rat models. RT-qPCR and Western blot analyses corroborated these findings, affirming the suppression of mammary hyperplasia by HZ-3 through the activation of estrogen pathway signaling.

Study on the solubilization effect of 7-ethyl-10-hydroxycamptothecin based on molecular docking and molecular dynamics simulation.

CONTEXT: With the continuous improvement of anticancer drugs, the condition of patients has been controlled to a certain extent, but the problem that still needs to be urgently solved is that most anticancer drug candidates' solubility is low. On the one hand, the low solubility of anticancer drugs may lead to a decrease in the absorption rate of anticancer drugs, poor treatment effect, and even death in severe cases. On the other hand, it will also lead to a waste of medical resources. At the same time, the rapid and scientific screening of ideal anticancer drugs has become a difficult problem that researchers have to face in the research process. In this study, we found two kinds of SN38-ligand complexes that solubilize 7-ethyl-10-hydroxycamptothecin (SN38) through molecular docking and molecular dynamics simulation methods. This process not only provided valuable information on improving the solubility of SN38, but also helped to discover effective potential complexes that solubilize SN38 quickly and scientifically. METHODS: The interaction of the SN38 with folic acid and isoproterenol hydrochloride was rapidly determined by molecular docking and molecular dynamics simulation methods. We used Discovery Studio software to perform molecular docking. And then, we used Gromacs 2019.3 software to perform molecular dynamics, analyzing and comparing the hydrogen bonds, solvent-accessible surface areas, energies, and so on between SN38 and SN38-ligand complexes. And the force field adopted the Gromos 54a7.

Construction of QSAR model based on cysteine-containing dipeptides and screening of natural tyrosinase inhibitors.

Considering that natural products as tyrosinase inhibitors are considered to be safe, with little or no toxic side effects and friendly to the environment, it is urgent to develop a new recognition strategy for natural tyrosinase inhibitors. In current study, an integrated computational analysis was conducted on Cys-containing dipeptides with high tyrosinase inhibitory activity. Firstly, molecular fingerprint similarity (FS) clustering analysis was performed on the target molecule using machine learning. Secondly, genetic algorithm was used to construct two kinds of highly accurate QSAR models (R2  = .978 and .984, respectively) with Cys at C-terminal and N-terminal. Finally, three novel natural candidate inhibitors (NP1, NP2, NP3) were discovered using Molnatsim natural product cluster library, automated screening process and QSAR based on the maximum common substructure (MCS) algorithm, their IC50pre were 260.96, 3.37 and 0.05 µm/mol. Pharmacokinetic predictions showed that NP2 and NP3 had high Bioavailability Score (BS) and Gastrointestinal (GI) absorption, and molecular dynamics simulations further validated the stability of these novel natural candidate inhibitors in binding to tyrosinase. In conclusion, our results provide new ideas for discovering new activities of natural products, and provide an accurate QSAR model for developing novel tyrosinase inhibitors based on MCS Cys-containing dipeptides. PRACTICAL APPLICATIONS: Tyrosinase is related to the occurrence of diseases such as excessive melanin deposition such as freckles and chloasma, and studies have shown that neurodegeneration associated with Parkinson's disease and Huntington's disease is also related. In addition, enzymatic browning on the surface of fresh fruit and vegetable slices will shorten the shelf life and affect their quality. Therefore, screening, designing and developing efficient tyrosinase inhibitors is very important in the fields of medicine, cosmetics, food and so on.

Serum Level of Brain-Derived Neurotrophic Factor (BDNF) Associated with Depression in Patients with Rosacea: A Candidate Predictive Biomarker.

Background: The biomarker to predict the depression in patients with rosacea was absent. Objective: We aimed to explore the potential association between BDNF and depression in patients with rosacea, and also to determine whether serum BDNF level is a potential biomarker for identifying depression in patients with rosacea. Methods: The patients with rosacea, rosacea with depression and healthy control were included, clinical evaluation (DLQI, RSSs, BDI-II) and serum BDNF levels detection were performed on subjects, the comparisons and correlation analysis of the obtained data were performed. Results: In clinical evaluation, whether DLQI or RSSs, rosacea with depression group was significantly higher compared to rosacea group. Besides, we found the serum BDNF levels were lower in patients with rosacea and rosacea with depression compared to healthy controls, also in the rosacea with depression group, serum BDNF levels were lower than in rosacea patients. Whatever in rosacea or rosacea with depression group, the statistical significance of serum BDNF levels between the different subtypes like the ETR and PPR was not found. In further correlation analysis, we found no correlation between serum BDNF and RSSs in patients with rosacea whatever the subtype of ETR or PPR. Interestingly, we found a negative correlation between serum BDNF levels and BDI-II in rosacea with depression group, the decreased serum BDNF levels were associated with the increased BDI-II, also the ROC confirmed it can evaluate the depression in patients with rosacea. Conclusion: Serum BDNF level is a potential biomarker for identifying depression in patients with rosacea.

Effects of hsa-mir-145-5p on the Regulation of msln Expression in Colorectal Adenocarcinoma.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers in the world, and its incidence is increasing all over the world including China. In recent years, research data show that some miRNAs are differentially expressed in cancer tissues, and their expression is closely contributed with the prognosis of CRC. Microarray technology was used, and 179 miRNAs were screened out with significantly altered expression in CRC tissues compared with adjacent tissues. The expression of mir-145-5p in tumor tissues was 3.48 times lower than that in normal tissues. Using bioinformatics technology and network resource prediction, we found that mir-145-5p had a potential target gene relationship with msln gene. Then, qRT-PCR was used to validate the expression level of mir-145-5p and msln mRNA in CRC and paracancerous tissues. The results showed that msln mRNA was higher than in normal tissues, while mir-145-5p was lower, with statistically significant difference (P < 0.01, n = 3). Furthermore, the expression of msln protein in CRC and normal colorectal tissues was detected by protein mass spectrometry (MRM) (n = 3) and immunohistochemistry in a total case of 30 colorectal cancer tissues and normal tissues. Result showed that the positive expression of msln in CRC was higher than that in normal colorectal tissues, 1.38e-6 and 1.89e-6, respectively (P < 0.01, n = 3). Furthermore, in 48 h RTCA real-time monitoring experiment, mir-145-5p showed inhibitory effect on the proliferation of colo320 cells stimulated by msln. This study demonstrated that msln is a target gene of mir-145-5p in CRC. Besides, mir-145-5p negatively regulates the proliferation of CRC colo320 cells through downregulating msln gene expression in CRC colo320 cells.

Traditional Mongolian medicine (HHQG) attenuates CCl4-induced acute liver injury through inhibiting monocyte/macrophage infiltration via the p-P38/p-JNK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Honghua Qinggan 13 Flavor Pills (HHQG), whose Mongolian name is Guri Gumu-13, is a traditional Mongolian medicine, that was stated in the "Diagnosis and Treatment of Ming Medical Code". The HHQG has been included in the Mongolian Medicine Division of the Ministry of Health Drug Standards (1998 edition). Based on our clinical expertise, HHQG demonstrated satisfactory therapeutic effects in hepatitis and liver failure. However, the pharmacological effects and potential mechanisms of HHQG have not been investigated. AIM OF THE STUDY: In this study, we combined network pharmacology, transcriptomics, and molecular biology to detect the underlying mechanism for the effect of HHQG on acute liver injury in mice. MATERIALS AND METHODS: Network pharmacology was used to explore the pathways involved in the protective effect HHQG in acute liver injury. This effect was further verified by injecting carbon tetrachloride (CCl4; 10 mL/kg, i.p.) to induce acute liver injury in mice. Serum markers of liver injury, morphology, histology, and monocyte/macrophage infiltration in the liver tissue were investigated. Transcriptomics further defined the HHQG targets. Transwell analysis was performed to confirm that HHQG inhibited monocyte/macrophage RAW.264.7 infiltration. qPCR and Western blot were performed to explore the mechanism of action of HHQG. RESULTS: Network pharmacology showed that HHQG exerted anti-oxidative and anti-inflammatory effects and promoted metabolic effects against acute liver injury. Pretreatment of mice with HHQG significantly maintained their body weight and decreased serum tumor necrosis factor-alpha (TNF-α) levels induced by CCl4 treatment in vivo. Histopathological examination further confirmed that HHQG protected the liver cells from CCl4-induced damage. Importantly, HHQG significantly inhibited CCl4-induced monocyte/macrophage infiltration. Transcriptomic analysis revealed that HHQG significantly reduced the expression of chemokines and cell adhesion molecules. We determined that HHQG significantly downregulated the expression of the key chemokine (monocyte chemokine protein-1, CCL2) at the gene and protein levels. Further research showed that HHQG inhibited chemokine production in hepatocytes by inhibiting the p-P38 and p-JNK pathways, thereby reducing monocyte/macrophage infiltration. CONCLUSIONS: These combined data showed that HHQG alleviated acute liver injury in mice, and further verified that HHQG exerted protective effects by inhibiting the production of CCL2 and reducing the infiltration of monocyte/macrophage by inhibiting the p-P38 and p-JNK pathways.

Molecular Simulation Study on the Interaction between Tyrosinase and Flavonoids from Sea Buckthorn.

Isorhamnetin, kaempferol, myricetin, and quercetin are four kinds of secondary metabolites in sea buckthorn, which have a wide range of biological activities. Investigating their interactions with tyrosinase at the atomic level can improve the bioavailability of sea buckthorn. Both molecular docking and molecular dynamics simulation methods were employed to study the interactions of these ligands with tyrosinase. The results of molecular docking indicated that these four small molecules such as isorhamnetin, kaempferol, myricetin, and quercetin can all dock into the active center of tyrosinase, and by occupying the active site, they can prevent substrate binding, thereby reducing the catalytic activity of tyrosinase. Molecular dynamics simulation trajectory analysis showed that all tyrosinase-ligand complexes reach an equilibrium within 100 ns. In addition, quercetin has the lowest binding energy among these four ligands, and the complex with tyrosinase is the most stable. This study not only provides valuable information for improving the bioavailability of sea buckthorn but also contributes to the discovery of effective natural inhibitors of tyrosinase.

Suppression of cell death by the secretory form of N-terminal ERC/mesothelin.

ERC/mesothelin is highly expressed in malignant mesothelioma, pancreatic cancer, and ovarian cancer. It is cleaved to a 30 kDa N-terminal secretory form (N-ERC) and a 40 kDa C-terminal membranous form (C-ERC). Several functions have been reported for full-length ERC (full-ERC) and C-ERC/mesothelin, such as in cell adhesion and invasion, stimulation of cell proliferation, and the suppression of cell death. However, there have been no studies to date on the function of secretory N-ERC, despite the fact that it is abundantly secreted into the sera of mesothelioma patients. In this study, we investigated whether N-ERC could function as a secretory factor to stimulate tumor progression. Full-, N, or C-ERC was overexpressed in the human hepatocellular carcinoma cell line Huh7 that lacks endogenous expression of ERC/mesothelin. Changes in the rates of cell proliferation and cell death were determined, and the state of signal transducers was examined using various endpoints: total cell counts, trypan blue exclusion rate, BrdU incorporation rate, TUNEL assay, and the phosphorylation of ERK1/2 and Stat3. In cells overexpressing N-ERC, phosphorylation of ERK1/2 was enhanced and the rate of cell death decreased, leading to the increase of cell number. The culture medium containing the secretory N-ERC also had the activity to increase the number of cells. Our data suggested that one of the full-ERC functions reported previously was mediated by the secretory N-ERC.

Mesothelin (MSLN) promoter is hypomethylated in malignant mesothelioma, but its expression is not associated with methylation status of the promoter.

Gene methylation leads to malignant progression in some tumors. The mechanism by which mesothelin is expressed in malignant mesothelioma (MM) is not well understood. MM is histologically divided into 3 subtypes, that is, the epithelioid, sarcomatoid, and biphasic types, and it was shown that mesothelin expression was restricted to the epithelioid type and the epithelioid component of the biphasic type of MM. However, its regulatory mechanism of expression has not been clarified. Here, we studied the expression of mesothelin by immunohistochemistry along with the methylation status of 20 CpG sites in the promoter of the mesothelin gene (MSLN) in 118 lung specimens, including 39 MM, 41 lung carcinoma, 26 nonneoplastic pulmonary lesions, and 12 normal lung tissue samples by the methylation-sensitive single nucleotide primer extension technique. We confirmed that mesothelin was expressed in the epithelioid type and epithelioid component of the biphasic type of MM but neither in the sarcomatoid type nor sarcomatous component of the biphasic type. Surprisingly, the MSLN promoter was significantly hypomethylated in the MM cases regardless of its subtype, compared with the other pulmonary lesions and normal lung tissue samples. These findings suggested that hypomethylation of the MSLN promoter may be specifically associated with the formation of MM, regardless of its expression status, and that the expression of mesothelin protein was lost in the sarcomatoid type by some unknown posttranscriptional regulatory mechanism. We also identified 4 CpG sites, among the 20 sites studied, to be more specifically hypomethylated in MM cases.

Establishment of the enzyme-linked immunosorbent assay system to detect the amino terminal secretory form of rat Erc/Mesothelin.

By representational difference analysis, we previously identified the rat Erc (Expressed in renal carcinoma) gene that was more abundantly expressed in the renal carcinoma tissues of Eker rats than in the rat normal kidney. In this study, we raised antibodies against the amino-terminal portion of the rat Erc, and demonstrated the existence of a approximately 30-kDa secretory form in the supernatant of cultured cells derived from rat renal carcinoma. The enzyme-linked immunosorbent assay (ELISA) system using these antibodies detected high concentrations of this form in the sera of Eker rats bearing renal carcinomas, and in the sera of rats transplanted with mesothelioma cells. Mesothelin, a human homolog of the rat Erc, was recently reported to be a serum marker of malignant mesothelioma. The prognosis of mesothelioma is poor and there is no effective treatment at present. There are several rat model systems of mesothelioma that may be promising tools in the development of an antimesothelioma treatment. We hope our ELISA to detect the soluble form of rat Erc/Mesothelin is useful in the rat model system to exploit the antimesothelioma therapy to be used in human cases.