Rapid Reduction of CEA and Stable Metastasis in an NRAS-mutant Rectal-Cancer Patient Treated With FOLFIRI and Bevacizumab Combined With Oral Recombinant Methioninase and a Low-Methionine Diet Upon Metastatic Recurrence After FOLFIRI and Bevacizumab Treatment Alone.

BACKGROUND/AIM: The choice of chemotherapy agents for RAS-mutant colorectal cancer is limited, and prognosis is poor compared to RAS-wild-type colorectal cancer. The purpose of the present study was to evaluate the effectiveness of methionine restriction combined with chemotherapy in a patient with NRAS-mutant rectal cancer. PATIENTS AND METHODS: A 59-year-old female was diagnosed with lung-metastatic recurrence of NRAS-mutant rectal cancer two and a half years after resection of the primary tumor. She started chemotherapy, which consisted of fluorouracil, irinotecan (FOLFIRI), and bevacizumab, in October 2020. Eight months later, stereotactic body radiation therapy (SBRT) was performed to treat the lung metastases. She stopped chemotherapy at this point and had blood tests and computed tomography (CT) scans regularly. Her CEA level increased to 139.91 ng/ml and her lung metastasis became larger by September 2022. Therefore, she was reintroduced to FOLFIRI and bevacizumab in October 2022, and also started a low-methionine diet and oral recombinant methioninase (o-rMETase) as a supplement. RESULTS: After starting the combination therapy with o-rMETase, a low-methionine diet, FOLFIRI, and bevacizumab, blood CEA levels very rapidly decreased and were almost within the normal limits five months later. CT findings showed the lung metastasis did not grow. CONCLUSION: Methionine restriction comprising o-rMETase and a low-methionine diet combined with first-line chemotherapy was effective in a patient with NRAS-mutant rectal cancer in which metastasis had re-occurred after first-line chemotherapy alone.

A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma.

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.

Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL.

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Current treatment options for treating OPA1-mutant dominant optic atrophy.

Dominant optic atrophy (DOA) is caused by OPA1 gene mutation, and it represents one of the most frequently diagnosed forms of hereditary optic neuropathies. This neurodegenerative disorder typically occurs in the first decades of life, and it is often associated with severe visual impairment. For this reason, several treatment options have been examined for the management of DOA, including vitamin supplements, ubiquinone analogues (in particular idebenone) and, more recently, gene therapy. Among them, idebenone has shown the most promising clinical outcomes in recent real-life studies. Furthermore, gene therapy represents also a promising therapeutic approach; however, more evidence in clinical trials is needed. In this review, we will summarize and discuss all the possible treatment options for DOA, in order to identify the current optimal management in these patients, whose visual prognosis remains unfortunately poor and unsatisfactory in the everyday clinical practice.

BRAF activation by metabolic stress promotes glycolysis sensitizing NRASQ61-mutated melanomas to targeted therapy.

NRAS-mutated melanoma lacks a specific line of treatment. Metabolic reprogramming is considered a novel target to control cancer; however, NRAS-oncogene contribution to this cancer hallmark is mostly unknown. Here, we show that NRASQ61-mutated melanomas specific metabolic settings mediate cell sensitivity to sorafenib upon metabolic stress. Mechanistically, these cells are dependent on glucose metabolism, in which glucose deprivation promotes a switch from CRAF to BRAF signaling. This scenario contributes to cell survival and sustains glucose metabolism through BRAF-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2/3 (PFKFB2/PFKFB3). In turn, this favors the allosteric activation of phosphofructokinase-1 (PFK1), generating a feedback loop that couples glycolytic flux and the RAS signaling pathway. An in vivo treatment of NRASQ61 mutant melanomas, including patient-derived xenografts, with 2-deoxy-D-glucose (2-DG) and sorafenib effectively inhibits tumor growth. Thus, we provide evidence for NRAS-oncogene contributions to metabolic rewiring and a proof-of-principle for the treatment of NRASQ61-mutated melanoma combining metabolic stress (glycolysis inhibitors) and previously approved drugs, such as sorafenib.

Successful treatment with MEK-inhibitor in a patient with NRAS-related cutaneous skeletal hypophosphatemia syndrome.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is caused by somatic mosaic NRAS variants and characterized by melanocytic/sebaceous naevi, eye, and brain malformations, and FGF23-mediated hypophosphatemic rickets. The MEK inhibitor Trametinib, acting on the RAS/MAPK pathway, is a candidate for CSHS therapy. A 4-year-old boy with seborrheic nevus, eye choristoma, multiple hamartomas, brain malformation, pleural lymphangioma and chylothorax developed severe hypophosphatemic rickets unresponsive to phosphate supplementation. The c.182A > G;p.(Gln61Arg) somatic NRAS variant found in DNA from nevus biopsy allowed diagnosing CSHS. We administered Trametinib for 15 months investigating the transcriptional effects at different time points by whole blood RNA-seq. Treatment resulted in prompt normalization of phosphatemia and phosphaturia, catch-up growth, chylothorax regression, improvement of bone mineral density, reduction of epidermal nevus and hamartomas. Global RNA sequencing on peripheral blood mononucleate cells showed transcriptional changes under MEK inhibition consisting in a strong sustained downregulation of signatures related to RAS/MAPK, PI3 kinase, WNT and YAP/TAZ pathways, reverting previously defined transcriptomic signatures. CSHS was effectively treated with a MEK inhibitor with almost complete recovery of rickets and partial regression of the phenotype. We identified "core" genes modulated by MEK inhibition potentially serving as surrogate markers of Trametinib action.

Genomic landscape of lymphatic malformations: a case series and response to the PI3Kα inhibitor alpelisib in an N-of-1 clinical trial.

Background: Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases. Methods: We examined the genomic landscape of a patient cohort of LMs (n = 30 cases) that underwent comprehensive genomic profiling using a large-panel next-generation sequencing assay. Immunohistochemical analyses were completed in parallel. Results: These LMs had low mutational burden with hotspot PIK3CA mutations (n = 20) and NRAS (n = 5) mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal LM harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable radiologic complete response, as defined in RECIST1.1, to the PI3Kα inhibitor alpelisib within the context of a personalized N-of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive to alpelisib treatment in vitro, which was demonstrated by a concentration-dependent drop in measurable impedance, an assessment of cell status. Conclusions: Our findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations. Funding: R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953. Clinical trial number: NCT03941782.

Research on Mfn2 Gene Expression in Hepatocellular Carcinoma and its Antitumor Mechanism.

Objective: To detect the expression level of the Mfn2 gene in hepatocellular carcinoma (HCC) and adjacent normal liver tissues and further analyze its anticancer effects. Methods: The expression levels of Mfn2, GLS1 and the autophagy-related proteins lc3b and Beclin1 in liver cancer and adjacent tissues in patients with liver cancer were detected by real-time-quantitative polymerase chain reaction (RT-qPCR). The HepG2 human HCC cell line was cultured in vitro, and the Mfn2 protein was stably expressed through transfection of a high Mfn2 expression plasmid. The Cell-Counting Kit-8 (CCK-8) method was used to observe the effect of Mfn2 overexpression on the activity of HepG2 cells. Furthermore, RT-qPCR and Western blotting were performed to detect the effects of Mfn2 overexpression on the protein expression of GLS1, Beclin1 and lc3b. Results: Compared with tissues adjacent to cancer tissues, the mRNA levels of Mfn2, GLS1, Beclin1 and lc3b in liver cancer tissues were lower. Compared with normal hepatocytes, the expression of Mfn2, Beclin1 and lc3b in HCC cells was decreased, but the expression of GLS1 was increased. Compared with the control group (NC) transfected with empty plasmid, Mfn2 overexpression led to significant time-dependent inhibition of HepG2 cell activity and GLS1 protein expression (P < .05). In addition, Mfn2 overexpression induced autophagy by triggering the expression of autophagy-related proteins Beclin-1 and lc3b in HCC cells (all P < .05). The effect of transfection with a high-dose Mfn2 plasmid was more obvious than that of transfection with a low-dose Mfn2 plasmid (all P < .05). Conclusions: The expression of Mfn2, GLS1, Beclin1 and lc3b in HCC was lower than in normal liver tissue. The expression of Mfn2, Beclin1 and lc3b in HCC cells was decreased, but the expression of GLS1 was increased. Overexpression of Mfn2 inhibited GLS1 gene expression by inhibiting the activity of HCC cells and promoted the expression of Beclin1 and lc3b to induce autophagy, thereby exerting an anticancer effect. Further research is needed to clarify the mechanism of Mfn2 activity.

Nicotinamide riboside promotes Mfn2-mediated mitochondrial fusion in diabetic hearts through the SIRT1-PGC1α-PPARα pathway.

Myocardial dysfunction is associated with an imbalance in mitochondrial fusion/fission dynamics in patients with diabetes. However, effective strategies to regulate mitochondrial dynamics in the diabetic heart are still lacking. Nicotinamide riboside (NR) supplementation ameliorated mitochondrial dysfunction and oxidative stress in both cardiovascular and aging-related diseases. This study investigated whether NR protects against diabetes-induced cardiac dysfunction by regulating mitochondrial fusion/fission and further explored the underlying mechanisms. Here, we showed an evident decrease in NAD+ (nicotinamide adenine dinucleotide) levels and mitochondrial fragmentation in the hearts of leptin receptor-deficient diabetic (db/db) mouse models. NR supplementation significantly increased NAD+ content in the diabetic hearts and promoted mitochondrial fusion by elevating Mfn2 level. Furthermore, NR-induced mitochondrial fusion suppressed mitochondrial H2O2 and O2•- production and reduced cardiomyocyte apoptosis in both db/db mice hearts and neonatal primary cardiomyocytes. Mechanistically, chromatin immunoprecipitation (ChIP) and luciferase reporter assay analyses revealed that PGC1α and PPARα interdependently regulated Mfn2 transcription by binding to its promoter region. NR treatment elevated NAD+ levels and activated SIRT1, resulting in the deacetylation of PGC1α and promoting the transcription of Mfn2. These findings suggested the promotion of mitochondrial fusion via oral supplementation of NR as a potential strategy for delaying cardiac complications in patients with diabetes.

Total flavonoids of Selaginella tamariscina (P.Beauv.) Spring ameliorates doxorubicin-induced cardiotoxicity by modulating mitochondrial dysfunction and endoplasmic reticulum stress via activating MFN2/PERK.

BACKGROUND: Doxorubicin (DOX) is a highly effective chemotherapeutic that is effective for various tumours. However, the clinical application of DOX has been limited by adverse reactions such as cardiotoxicity and heart failure. Since DOX-induced cardiotoxicity is irreversible, drugs to prevent DOX-induced cardiotoxicity are needed. PURPOSE: This study aimed to investigate the effect of total flavonoids of Selaginella tamariscina (P.Beauv.) Spring (TFST) on doxorubicin-induced cardiotoxicity. METHODS: The present study established DOX-induced cardiotoxicity models in C57BL/6 mice treated with DOX (cumulative dose: 20 mg/kg body weight) and H9c2 cells incubated with DOX (1 µM/l) to explore the intervention effect and potential mechanism of TFST. Echocardiography was performed to evaluate left ventricular functions. Heart tissue samples were collected for histological evaluation. Myocardial injury markers and oxidative stress markers were examined. Mitochondrial energy metabolism pathway associated proteins PPARα/PGC-1α/Sirt3 were detected. We also explored the effects of TFST on endoplasmic reticulum (ER) stress and apoptosis. To further investigate the protective mechanism of TFST, we used the specific small interfering RNA MFN2 (siMFN2) to explore the effect of MFN2 on TFST against DOX-induced cardiotoxicity in vitro. Flow cytometry detected reactive oxygen species, mitochondrial membrane potential and apoptosis. Cell mitochondrial stress was measured by Seahorse XF analyser. RESULTS: Both in vivo and in vitro studies verified that TFST observably alleviated DOX-induced mitochondrial dysfunction and ER stress. However, these effects were reversed after transfected siMFN2. CONCLUSION: Our results indicated that TFST ameliorates DOX-induced cardiotoxicity by alleviating mitochondrial dysfunction and ER stress by activating MFN2/PERK. MFN2/PERK pathway activation may be a novel mechanism to protect against DOX-induced cardiotoxicity.

Arabidopsis pavement cell morphogenesis requires FERONIA binding to pectin for activation of ROP GTPase signaling.

Sensing and signaling of cell wall status and dynamics regulate many processes in plants, such as cell growth and morphogenesis, but the underpinning mechanisms remain largely unknown. Here, we demonstrate that the CrRLK1L receptor kinase FERONIA (FER) binds the cell wall pectin, directly leading to the activation of the ROP6 guanosine triphosphatase (GTPase) signaling pathway that regulates the formation of the puzzle piece shape of pavement cells in Arabidopsis. The extracellular malectin domain of FER binds demethylesterified pectin in vivo and in vitro. Both loss-of-FER mutations and defects in pectin demethylesterification caused similar changes in pavement cell shape and ROP6 GTPase signaling. FER is required for the activation of ROP6 by demethylesterified pectin and physically and genetically interacts with the ROP6 activator, RopGEF14. Thus, our findings elucidate a signaling pathway that directly connects the cell wall pectin to cellular morphogenesis via the cell surface receptor FER.

Mechano-transduction via the pectin-FERONIA complex activates ROP6 GTPase signaling in Arabidopsis pavement cell morphogenesis.

During growth and morphogenesis, plant cells respond to mechanical stresses resulting from spatiotemporal changes in the cell wall that bear high internal turgor pressure. Microtubule (MT) arrays are reorganized to align in the direction of maximal tensile stress, presumably reinforcing the local cell wall by guiding the synthesis of cellulose. However, how mechanical forces regulate MT reorganization remains largely unknown. Here, we demonstrate that mechanical signaling that is based on the Catharanthus roseus RLK1-like kinase (CrRLK1L) subfamily receptor kinase FERONIA (FER) regulates the reorganization of cortical MT in cotyledon epidermal pavement cells (PCs) in Arabidopsis. Recessive mutations in FER compromised MT responses to mechanical perturbations, such as single-cell ablation, compression, and isoxaben treatment, in these PCs. These perturbations promoted the activation of ROP6 guanosine triphosphatase (GTPase) that acts directly downstream of FER. Furthermore, defects in the ROP6 signaling pathway negated the reorganization of cortical MTs induced by these stresses. Finally, reduction in highly demethylesterified pectin, which binds the extracellular malectin domains of FER and is required for FER-mediated ROP6 activation, also impacted mechanical induction of cortical MT reorganization. Taken together, our results suggest that the FER-pectin complex senses and/or transduces mechanical forces to regulate MT organization through activating the ROP6 signaling pathway in Arabidopsis.

Omega-3 fatty acids promote neuroprotection, decreased apoptosis and reduced glial cell activation in the retina of a mouse model of OPA1-related autosomal dominant optic atrophy.

The purpose of this study was to evaluate the neuroprotective effects of omega-3 polyunsaturated fatty acid (ω3-PUFA) supplementation in a mouse model of OPA1-associated autosomal dominant optic atrophy (ADOA). The blood level of arachidonic acid (AA) and eicosapentaenoic acid (EPA) served to adjust the treatment dosage (AA/EPA = 1.0-1.5). Eight-month-old mice were allocated to four groups (n = 20/group): the ω3-PUFA-treated Opa1enu/+, untreated Opa1enu/+, ω3-PUFA-treated wild-type and untreated wild-type groups. Treated mice received the ω3-PUFAs, EPA and docosahexaenoic acid (DHA; 5:1 ratio) by daily gavage for 4 months based on the measured AA/EPA ratio. Blood, retina and optic nerve (ON) fatty acid levels were determined by gas chromatography, and the retina and ON were histologically examined. Western blotting and/or immunohistochemistry was performed to analyse retinal mediators involved in Opa1-mutation-mediated apoptosis, inflammation and oxidative stress. Increased EPA and reduced AA levels were primarily observed predominantly in the blood and retinal tissues, and a similarly high EPA level tended to be observed in the ONs of ω3-PUFA-treated mice. Retinal ganglion cell and ON axonal densities were higher in both mouse strains upon ω3-PUFA treatment than in the corresponding untreated groups. Caspase-3 expression analysis showed fewer apoptotic retinal cells in both groups of treated mice. Decreases in inflammatory microglia and astrocytes activation and proapoptotic Bcl-2-associated X protein (Bax) expression were noted in the treated groups, with no difference in the antioxidant superoxide dismutase-2 expression. ω3-PUFA supplementation had neuroprotective effects on the retinas of Opa1enu/+ and wild-type mice via blockade of microglia and astrocytes activation and suppression of Bax and caspase-3. Our findings indicated that inhibition of oxidative stress may not be involved in ω3-PUFA-mediated neuroprotection. These novel findings support the use of ω3-PUFAs as a beneficial therapy in the occurrence of ADOA, posing the basis for future clinical trials to confirm these observations.

Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development.

Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria-targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER-located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca2+ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2-dependent ER-mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER-targeted Mfn2 or an artificial ER-mitochondria tether, indicating that manipulation of ER-mitochondria contacts could be used to treat pathologic conditions involving Mfn2.

Molecularly profiled trials: toward a framework of actions for the "nil actionables".

The sequencing of tumour or blood samples is increasingly used to stratify patients into clinical trials of molecularly targeted agents, and this approach has frequently demonstrated clinical benefit for those who are deemed eligible. But what of those who have no clear and evident molecular driver? What of those deemed to have "nil actionable" mutations? How might we deliver better therapeutic opportunities for those left behind in the clamour toward stratified therapeutics? And what significant learnings lie hidden in the data we amass but do not interrogate and understand? This Perspective article suggests a holistic approach to the future treatment of such patients, and sets a framework through which significant additional patient benefit might be achieved. In order to deliver upon this framework, it encourages and invites the clinical community to engage more enthusiastically and share learnings with colleagues in the early drug discovery community, in order to deliver a step change in patient care.

Exploring the effect of Gupi Xiaoji Prescription on hepatitis B virus-related liver cancer through network pharmacology and in vitro experiments.

AIM AND OBJECTIVE: To study the effect of Gupi Xiaoji Prescription (GXP) on hepatitis B virus(HBV)-related liver cancer through network pharmacology coupled with in vitro experiments and explore their related mechanisms. MATERIALS AND METHODS: Gupi Xiaoji Prescription's chemical constituents and the action targets of its six medicinal components were identified using several databases. These included the Traditional Chinese Medicine Systems Pharmacology Database （TCMSP), the Bioinformatics Analysis Tool for Molecular mechANism of TCM (BATMAN-TCM), and the Traditional Chinese Medicine Integrated Database (TCMID), while GeneCards and OMIM were used to compile relevant liver cancer disease targets. Pathway enrichment of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), analysis of potential targets, and analysis of the enriched pathways in literature were executed in R. The Hepatocellular carcinoma (HCC)-derived HepG2.2.15 cell line stably expresses and replicates HBV. In vitro experiments with HepG2.2.15 were used to verify GXP's effects on HBV-related liver cancer, while the human liver cancer cell line HepG2 was used as the control. RESULTS: 171 active ingredients and 259 potential drug targets were screened from GXP, involving 181 pathways in vitro. These assays identified Polyphyllin I as an effective GXP component. Notably, GXP inhibited cell proliferation and metastasis in a concentration-dependent manner (P < 0.01). In comparison with the vehicle group, the fluorescence intensity of each drug group was significantly weakened (P < 0.01), while the drug group Mitofusins 1(MFN1) and protein expression level of Mitofusins 2 (MFN2) increased significantly. The protein expression level of Mitochondrial fission protein 1 (FIS1) and Optic Atrophy 1 (OPA1) also showed significant decreases (P < 0.01). Molecular docking revealed Fructus saponins I's high affinity with FIS1, MFN1, MFN2, and OPA1. CONCLUSION: The network pharmacology results indicate that Gupi Xiaoji Prescription may treat liver cancer by regulating mitochondrial division and fusion of key genes to disrupt liver cancer cells' energy metabolism. In vitro experiments also verified that GXP could inhibit the proliferation and migration of HepG2.2.15 cells by up-regulating MFN1 and MFN2, down-regulating the expression of FIS1 and OPA1 in addition to damaging mitochondria. Consistent with network pharmacology and molecular docking results, Polyphyllin I may be the most active compound of the formula's components. It also shows that Traditional Chinese medicine (TCM) plays a significant, targeted role in the treatment of HBV-related liver cancer.

Exposure to oxidized soybean oil induces mammary mitochondrial injury in lactating rats and alters the intestinal barrier function of progeny.

Similar to other food contaminants, dietary oxidized soybean oil (OSO) is also a toxic xenobiotic for animal and human nutrition. This research evaluated the effects of maternal OSO exposure during lactation on mammary mitochondrial injury and intestinal barrier of sucking progeny. Twenty-four female adult SD rats were fed a fresh soybean oil (FSO) homozygous diet (7%) or an OSO homozygous diet (7%) during lactation. On day 21 of lactation, upregulated mRNA expression of Sirt3 and PRDX3 and downregulated mRNA expression of Mfn2 were observed in mammary tissues in the OSO group compared to the control group (P < 0.05). Maternal OSO consumption increased the FasL transcriptional level in the mammary glands of rat dams (P < 0.05), while the mRNA expression of Bax, Bcl-2, Caspase3, and Fas was not different from that in the control group (P > 0.05). OSO enhanced the Nrf2 transcriptional level and decreased the expression of Keap1 and PPARα in mammary tissues (P < 0.05). In addition, the contents of CAT, MDA, SOD were not affected by dietary OSO (P > 0.05), while the concentration of H2O2 was significantly decreased in the OSO-treated mammary glands of rat dams (P < 0.05). Maternal OSO exposure during lactation did not affect the organ coefficients of pups (P > 0.05). However, maternal OSO consumption influenced the intestinal tight junction protein expression of progeny (P < 0.05). In summary, the present study demonstrated that dietary OSO may aggravate mammary injury and mitochondria dysfunction, but the OSO-induced damage was self-alleviating via the promotion of Sirt3 and PRDX3 expression and further scavenging of oxidative products.

Secondary CoQ10 deficiency, bioenergetics unbalance in disease and aging.

Coenzyme Q10 (CoQ10 ) deficiency is a rare disease characterized by a decreased accumulation of CoQ10 in cell membranes. Considering that CoQ10 synthesis and most of its functions are carried out in mitochondria, CoQ10 deficiency cases are usually considered a mitochondrial disease. A relevant feature of CoQ10 deficiency is that it is the only mitochondrial disease with a successful therapy available, the CoQ10 supplementation. Defects in components of the synthesis machinery caused by mutations in COQ genes generate the primary deficiency of CoQ10 . Mutations in genes that are not directly related to the synthesis machinery cause secondary deficiency. Cases of CoQ10 deficiency without genetic origin are also considered a secondary deficiency. Both types of deficiency can lead to similar clinical manifestations, but the knowledge about primary deficiency is deeper than secondary. However, secondary deficiency cases may be underestimated since many of their clinical manifestations are shared with other pathologies. This review shows the current state of secondary CoQ10 deficiency, which could be even more relevant than primary deficiency for clinical activity. The analysis covers the fundamental features of CoQ10 deficiency, which are necessary to understand the biological and clinical differences between primary and secondary CoQ10 deficiencies. Further, a more in-depth analysis of CoQ10 secondary deficiency was undertaken to consider its origins, introduce a new way of classification, and include aging as a form of secondary deficiency.

Lathyrus sativus diamine oxidase reduces Clostridium difficile toxin A-induced toxicity in Caco-2 cells by rescuing RhoA-GTPase and inhibiting pp38-MAPK/NF-κB/HIF-1α activation.

Clostridium difficile toxin A (TcdA) impairs the intestinal epithelial barrier, increasing the mucosa permeability and triggering a robust inflammatory response. Lathyrus sativus diamino oxidase (LSAO) is a nutraceutical compound successfully used in various gastrointestinal dysfunctions. Here, we evaluated the LSAO (0.004-0.4 µM) ability to counter TcdA-induced (30 ng/mL) toxicity and damage in Caco-2 cells, investigating its possible mechanism of action. LSAO has improved the transepithelial electrical resistance (TEER) score and increased cell viability in TcdA-treated cells, significantly rescuing the protein expression of Ras homolog family members, A-GTPase (RhoA-GTPase), occludin, and zonula occludens-1 (ZO-1). LSAO has also exhibited an anti-apoptotic effect by inhibiting the TcdA-induced expression of Bcl-2-associated X protein (Bax), p50 nuclear factor-kappa-B (p50), p65nuclear factor-kappa-B (p65), and hypoxia-inducible transcription factor-1 alpha (HIF-1α), and the release of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF) in the cell milieu. Our data showed that LSAO exerts a protective effect on TcdA-induced toxicity in Caco-2 cells, placing itself as an interesting nutraceutical to supplement the current treatment of the Clostridium difficile infections.