Bioinformatics approach and experimental validation reveal the hepatoprotective effect of pachyman against acetaminophen-associated liver injury.

Pachyman, known as Poria cocos polysaccharides, refers to the bioactive compounds isolated from Poria cocos. Pachyman is thought to exert cytoprotective action. However, the detailed mechanisms of pachyman action for hepatoprotection remain unknown. In this study, we aimed to assess the therapeutic actions, molecular mechanisms, and key target proteins of pachyman in the treatment of liver injury through network pharmacology and molecular docking assays. Furthermore, these bioinformatic findings were validated by an acetaminophen (APAP)-induced liver injury in vivo. Primarily using bioinformatic analysis, we screened and characterized 12 genes that act as potential therapeutic targets of pachyman against APAP-induced liver injury, in which all core targets were obtained. By using enrichment analysis, these core target genes of pachyman were characterized to reveal the pharmacological functions and molecular mechanisms of anti-liver injury induced by APAP. A molecular docking simulation was further performed to certain anti-liver injury target proteins of pachyman, including cytochrome P450 3A4 enzyme (CYP3A4) and inducible nitric oxide synthase (NOS2). In animal experiments, pachyman exerted potent hepatoprotective activities in prenatal APAP-exposed offspring livers, characterized by activated hepatocellular CYP3A4 and NOS2 expressions. These current findings have thus indicated that pachyman exerts hepatoprotective effects and may be the promising nutraceuticals for the treatment of APAP-induced liver injury.

Identification and validation of a novel cuproptosis-related stemness signature to predict prognosis and immune landscape in lung adenocarcinoma by integrating single-cell and bulk RNA-sequencing.

Background: Cancer stem cells (CSCs) play vital roles in lung adenocarcinoma (LUAD) recurrence, metastasis, and drug resistance. Cuproptosis has provided a novel insight into the treatment of lung CSCs. However, there is a lack of knowledge regarding the cuproptosis-related genes combined with the stemness signature and their roles in the prognosis and immune landscape of LUAD. Methods: Cuproptosis-related stemness genes (CRSGs) were identified by integrating single-cell and bulk RNA-sequencing data in LUAD patients. Subsequently, cuproptosis-related stemness subtypes were classified using consensus clustering analysis, and a prognostic signature was constructed by univariate and least absolute shrinkage operator (LASSO) Cox regression. The association between signature with immune infiltration, immunotherapy, and stemness features was also investigated. Finally, the expression of CRSGs and the functional roles of target gene were validated in vitro. Results: We identified six CRSGs that were mainly expressed in epithelial and myeloid cells. Three distinct cuproptosis-related stemness subtypes were identified and associated with the immune infiltration and immunotherapy response. Furthermore, a prognostic signature was constructed to predict the overall survival (OS) of LUAD patients based on eight differently expressed genes (DEGs) with cuproptosis-related stemness signature (KLF4, SCGB3A1, COL1A1, SPP1, C4BPA, TSPAN7, CAV2, and CTHRC1) and confirmed in validation cohorts. We also developed an accurate nomogram to improve clinical applicability. Patients in the high-risk group showed worse OS with lower levels of immune cell infiltration and higher stemness features. Ultimately, further cellular experiments were performed to verify the expression of CRSGs and prognostic DEGs and demonstrate that SPP1 could affect the proliferation, migration, and stemness of LUAD cells. Conclusion: This study developed a novel cuproptosis-related stemness signature that can be used to predict the prognosis and immune landscape of LUAD patients, and provided potential therapeutic targets for lung CSCs in the future.

Active post-transcriptional regulation and ACLY-mediated acetyl-CoA synthesis as a pivotal target of Shuang-Huang-Sheng-Bai formula for lung adenocarcinoma treatment.

BACKGROUND: New therapeutic approaches are required to improve the outcomes of lung cancer (LC), a leading cause of cancer-related deaths worldwide. Chinese herbal medicine formulae widely used in China provide a unique opportunity for improving LC treatment, and the Shuang-Huang-Sheng-Bai (SHSB) formula is a typical example. However, the underlying mechanisms of action remains unclear. PURPOSE: This study aimed to confirm the efficacy of SHSB against lung adenocarcinoma (LUAD), which is a major histological type of LC, unveil the downstream targets of this formula, and assess the clinical relevance and biological roles of the newly identified target. METHODS: An experimental metastasis mouse model and a subcutaneous xenograft mouse model were used to evaluate the anti-cancer activity of SHSB. Multi-omics profiling of subcutaneous tumors and metabolomic profiling of sera were performed to identify downstream targets, especially the metabolic targets of SHSB. A clinical trial was conducted to verify the newly identified metabolic targets in patients. Next, the metabolites and enzymes engaged in the metabolic pathway targeted by SHSB were measured in clinical samples. Finally, routine molecular experiments were performed to decipher the biological functions of the metabolic pathways targeted by SHSB. RESULTS: Oral SHSB administration showed overt anti-LUAD efficacy as revealed by the extended overall survival of the metastasis model and impaired growth of implanted tumors in the subcutaneous xenograft model. Mechanistically, SHSB administration altered protein expression in the post-transcriptional layer and modified the metabolome of LUAD xenografts. Integrative analysis demonstrated that SHSB markedly inhibited acetyl-CoA synthesis in tumors by post-transcriptionally downregulating ATP-citrate lyase (ACLY). Consistently, our clinical trial showed that oral SHSB administration declined serum acetyl-CoA levels of patients with LC. Moreover, acetyl-CoA synthesis and ACLY expression were both augmented in clinical LUAD tissues of patients, and high intratumoral ACLY expression predicted a detrimental prognosis. Finally, we showed that ACLY-mediated acetyl-CoA synthesis is essential for LUAD cell growth by promoting G1/S transition and DNA replication. CONCLUSION: Limited downstream targets of SHSB for LC treatment have been reported in previous hypothesis-driven studies. In this study, we conducted a comprehensive multi-omics investigation and demonstrated that SHSB exerted its anti-LUAD efficacy by actively and post-transcriptionally modulating protein expression and particularly restraining ACLY-mediated acetyl-CoA synthesis.

Vitamin C protects the spleen against PFOA-induced immunotoxicity.

Perfluorooctanoic acid (PFOA) is widely used in industrial and consumer products of our daily life. It is well-documented that PFOA is closely associated with fatty liver disease. Recently, cumulating studies demonstrated the immunotoxicity of PFOA, but its harmful effect on the largest immune organ, spleen is still largely unknown. In the present study, we used PFOA-exposed mouse model together with comparative transcriptomic analysis to understand the molecular mechanisms underlying the immunotoxicity of PFOA. Furthermore, we investigated the possible use of vitamin C to reverse the PFOA-induced immunotoxicity in spleen. Our result showed that the PFOA exposure could reduce the spleen weight and plasma lymphocytes, and the splenic comparative transcriptomic analysis highlighted the alteration of cell proliferation, metabolism and immune response through the regulation of gene clusters including nicotinamide nucleotide transhydrogenases (NNT) and lymphocyte antigen 6 family member D and K (LY6D and LY6K). More importantly, the supplementation of vitamin C would relieve the PFOA-reduced spleen index and white blood cells. The bioinformatic analysis of transcriptome suggested its involvement in the spleen cell proliferation and immune response. For the first time, our study delineated the molecular mechanisms underlying the PFOA-induced immunotoxicity in the spleen. Furthermore, our results suggested that the supplementation of vitamin C had beneficial effect on the PFOA-altered spleen functions.

Exosomal lncRNA HOTAIR promotes osteoclast differentiation by targeting TGF-ß/PTHrP/RANKL pathway.

Bone tissue is a common metastatic site of lung cancer, and bone metastasis is characterized by abnormal differentiation and malfunction of osteoclast, and the roles of exosomes derived from lung cancer have attracted much attention. In our study, we found that the level of HOTAIR expression in A549 and H1299 exosomes was higher than those of normal lung fibrocytes. Overexpression of HOTAIR in A549 and H1299 exosomes promoted osteoclast differentiation. Furthermore, A549-Exos and H1299-Exos targeted bone tissues, and bone formation was significantly inhibited in vivo. Mechanistically, exosomal lncRNA HOTAIR promoted bone resorption by targeting TGF-ß/PTHrP/RANKL pathway.

Exosomal miR-328 originated from pulmonary adenocarcinoma cells enhances osteoclastogenesis via downregulating Nrp-2 expression.

Osseous metastases of pulmonary carcinoma and the detailed mechanisms remain unclear, and the effects of exosomes (Exos) originated from pulmonary adenocarcinoma cells in this process have received a lot of attentions. Our study revealed that the Exos secreted from A549 cells (A549-Exos) enhanced osteoclastogenesis and osseous resorption in vitro. In addition, A549-Exos showed a targeted effect on bones to enhance osseous resorption in vivo. A549-exosomal miR-328 enhanced osseous resorption via downregulating neuropilin 2 (Nrp-2) expression, and A549-Exos miR-328 inhibitors suppressed osseous resorption in vivo. Therefore, A549-exosomal miR-328 enhances osteoclastogenesis via downregulating Nrp-2 expression, thus A549-Exos miR-328 inhibitors can be used as a potential nanodrug for treating osseous metastases.

Bioinformatics and In Silico Findings Uncover Bio-Targets of Calycosin Against Heart Failure and Diabetes Mellitus.

Background: Heart failure (HF) and diabetes mellitus (DM) are life-threatening diseases. However, existing clinical drugs to treat HF complicated with DM are relatively limited. In this study, we performed a viable bioinformatics strategy combining network pharmacology and molecular docking to identify potential anti-HF and -DM targets and therapeutic mechanisms of calycosin, a functional phytoestrogen. Methods: Web-based databases were used to collect candidate genes/targets of calycosin and HF/DM and then identify the hub bio-targets of calycosin against HF/DM. Using the online-available database, all functional processes and signaling pathways of calycosin against HF/DM were screened and identified before further visualization. Results: All potential bio-targets of calycosin and HF/DM were collected, and 20 hub targets of calycosin against HF/DM were identified. Interestingly, molecular docking findings indicated that mitogen-activated protein kinase-1 (MAPK1), ß-arrestin 1 (ARRB1), and homologue-1 (ABL1) may be potent pharmacological targets of calycosin against HF/DM. In addition, all primary molecular functions of calycosin against HF/DM were identified, including regulating protein binding, ubiquitination, and the metabolic process. Furthermore, the top molecular pathways of calycosin against HF/DM were revealed, including cardiomyocyte and chemokine signaling pathways. Conclusion: Our bioinformatics analysis uncovered the network targets and therapeutic mechanisms of calycosin against HF/DM. For the first time, the current in silico findings revealed that the identified hub targets may be used to screen and treat HF/DM.

In†Silico Analysis Identifies the Anti-Liver Injury Targets of Diammonium Glycyrrhizinate: Validated in Perfluorooctanoic Acid-Lesioned Mouse Model.

Liver injury refers to a pathological condition that causes dysfunction to hepatic parenchymal cells. And diammonium glycyrrhizinate (DG) is clinically prescribed for hepatoprotection. To date, detailed information regarding DG against liver injury in molecular mechanisms remains unrevealed totally. In the present study, we applied network pharmacology and molecular docking to decipher substantial genes, biological functions of DG for treating liver injury. Furthermore, preclinical experiments using perfluorooctanoic acid (PFOA)-induced liver injury in mice were used to validate the bioinformatic findings. Our results showed that the target network of DG and liver injury predominantly shared 90 genes. Eleven core genes of DG treating liver injury including ALB, TP53, TNF, CASP3, PTGS2, JUN, TLR4, IL10, STAT3, NOS3, FOS. The gene ontology and KEGG enrichment further highlighted their importance in regulation of cell proliferation, regulation of transcription, inflammatory response, regulation of NF-kappaB import into nucleus, regulation of apoptotic process, T cell receptor signaling pathway, and Toll-like receptor signaling pathway. Moreover, DG treatment was found to rescue the PFOA-induced liver injury through the modulation of identified genes including TNF, CASP3, PTGS2, and ALB. Current integrated data from bioinformatics method and experimental validation uncovered that DG exerts potent actions to treat liver injury through regulating core targets associated with inflammation and immunomodulation.

Bioinformatics and in-silico findings reveal medical features and pharmacological targets of biochanin A against colorectal cancer and COVID-19.

Severe mortality due to the COVID-19 pandemic resulted from the lack of effective treatment. Although COVID-19 vaccines are available, their side effects have become a challenge for clinical use in patients with chronic diseases, especially cancer patients. In the current report, we applied network pharmacology and systematic bioinformatics to explore the use of biochanin A in patients with colorectal cancer (CRC) and COVID-19 infection. Using the network pharmacology approach, we identified two clusters of genes involved in immune response (IL1A, IL2, and IL6R) and cell proliferation (CCND1, PPARG, and EGFR) mediated by biochanin A in CRC/COVID-19 condition. The functional analysis of these two gene clusters further illustrated the effects of biochanin A on interleukin-6 production and cytokine-cytokine receptor interaction in CRC/COVID-19 pathology. In addition, pathway analysis demonstrated the control of PI3K-Akt and JAK-STAT signaling pathways by biochanin A in the treatment of CRC/COVID-19. The findings of this study provide a therapeutic option for combination therapy against COVID-19 infection in CRC patients.

Exploring anti-liver cancer targets and mechanisms of oxyresveratrol: in silico and verified findings.

The aim of current study was to exhume the potential targets and molecular mechanisms of oxyresveratrol, a structurally re-constructed resveratrol, for treating liver cancer through bioinformatics investigation and experimentative validation. To start with, the network pharmacology approach and molecular docking technology were used to uncover all candidate targets of oxyresveratrol to treat liver cancer, accompanied with identified anti-liver cancer targets including estrogen receptor 1 (ESR1), epidermal growth factor receptor (EGFR). In addition, more pharmacological mechanisms of oxyresveratrol against liver cancer were revealed in details. In experimental verification, the clinical samples of liver cancer showed elevated ESR1, EGFR mRNA expressions. The in-vitro data indicated that intracellular contents of ESR1, EGFR mRNAs in oxyresveratrol-treated liver cancer cells were reduced. Taken together, the bioinformatics and validated findings have highlighted detailed pharmacological targets and molecular mechanisms of oxyresveratrol for treating liver cancer. Following with experimental verification, the identified genes of ESR1, EGFR may function as potential screening anti-liver cancer markers.

Effect of Polyhydroxybutyrate (PHB) storage on L-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation.

BACKGROUND: Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-ß-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5. RESULTS: C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain. CONCLUSIONS: Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield L-arginine.