Deciphering the neuroprotective mechanisms of RACK1 in cerebral ischemia-reperfusion injury: Pioneering insights into mitochondrial autophagy and the PINK1/Parkin axis.

INTRODUCTION: Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. METHODS: We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. RESULTS: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. CONCLUSION: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.

RING finger protein 5 protects against acute myocardial infarction by inhibiting ASK1.

BACKGROUND: Myocardial infarction (MI) is a major disease with high morbidity and mortality worldwide. However, existing treatments are far from satisfactory, making the exploration of potent molecular targets more imperative. The E3 ubiquitin ligase RING finger protein 5 (RNF5) has been previously reported to be involved in several diseases by regulating ubiquitination-mediated protein degradation. Nevertheless, few reports have focused on its function in cardiovascular diseases, including MI. METHODS: In this study, we established RNF5 knockout mice through precise CRISPR-mediated genome editing and utilized left anterior descending coronary artery ligation in 9-11-week-old male C57BL/6 mice. Subsequently, serum biochemical analysis and histopathological examination of heart tissues were performed. Furthermore, we engineered adenoviruses for modulating RNF5 expression and subjected neonatal rat cardiomyocytes to oxygen-glucose deprivation (OGD) to mimic ischemic conditions, demonstrating the impact of RNF5 manipulation on cellular viability. Gene and protein expression analysis provided insights into the molecular mechanisms. Statistical methods were rigorously employed to assess the significance of experimental findings. RESULTS: We found RNF5 was downregulated in infarcted heart tissue of mice and NRCMs subjected to OGD treatment. RNF5 knockout in mice resulted in exacerbated heart dysfunction, more severe inflammatory responses, and increased apoptosis after MI surgery. In vitro, RNF5 knockdown exacerbated the OGD-induced decline in cell activity, increased apoptosis, while RNF5 overexpression had the opposite effect. Mechanistically, it was proven that the kinase cascade initiated by apoptosis signal-regulating kinase 1 (ASK1) activation was closely regulated by RNF5 and mediated RNF5's protective function during MI. CONCLUSIONS: We demonstrated the protective effect of RNF5 on myocardial infarction and its function was dependent on inhibiting the activation of ASK1, which adds a new regulatory component to the myocardial infarction associated network and promises to enable new therapeutic strategy.

Alendronate sodium induces G1 phase arrest and apoptosis in human umbilical vein endothelial cells by inhibiting ROS-mediated ERK1/2 signaling.

Bisphosphonates are potent bone resorption inhibitors, among which alendronate sodium (ALN) is commonly prescribed for most osteoporosis patients, but long-term application of ALN can cause bisphosphonate-related osteonecrosis of jaw (BRONJ), the pathogenesis of which remains unclear. Previous studies have suggested that bisphosphonates cause jaw ischemia by affecting the biological behavior of vascular endothelial cells, leading to BRONJ. However, the impacts of ALN on vascular endothelial cells and its mechanism remain unclear. The purpose of this work is to assess the influence of ALN on human umbilical vein endothelial cells (HUVECs) and clarify the molecular pathways involved. We found that high concentration of ALN induced G1 phase arrest in HUVECs, demonstrated by downregulation of Cyclin D1 and Cyclin D3. Moreover, high concentration of ALN treatment showed pro-apoptotic effect on HUVECs, demonstrated by increased levels of the cleaved caspase-3, the cleaved PARP and Bax, along with decreased levels of anti-apoptotic protein Bcl-2. Further experiments showed that ERK1/2 phosphorylation was decreased. Additionally, ALN provoked the build-up of reactive oxygen species (ROS) in HUVECs, leading to ERK1/2 pathway suppression. N-acetyl-L-cysteine (NAC), a ROS scavenger, efficiently promoted the ERK1/2 phosphorylation and mitigated the G1 phase arrest and apoptosis triggered by ALN in HUVECs. PD0325901, an inhibitor of ERK1/2 that diminishes the ERK1/2 phosphorylation enhanced the ALN-induced G1 phase arrest and apoptosis in HUVECs. These findings show that ALN induces G1 phase arrest and apoptosis through ROS-mediated ERK1/2 pathway inhibition in HUVECs, providing novel insights into the pathogenic process, prevention and treatment of BRONJ in individuals receiving extended use of ALN.

Ad-VT causes ovarian cancer A2780 cell death via mitochondrial apoptosis and autophagy pathways.

OBJECTIVE: The recombinant adenovirus Ad-apoptin-hTERTp-E1a (Ad-VT) to have a bi-specific oncolytic character in many tumor cells, but its action pathway in killing tumor cells has not been accurately elucidated. Here, we studied the mechanism of apoptosis and autophagy induced by Ad-VT and the interaction between autophagy and apoptosis. METHODS: Crystal Violet staining and CCK-8 assays were used to detect the inhibitory effect of Ad-VT on ovarian cancer cells. The antitumor effect of Ad-VT in vivo was analyzed by tumor bearing nude mouse model. Subsequently, flow cytometry and fluorescence staining were used to analyze the main types of apoptosis and autophagy induced by Ad-VT. RESULTS: In this study, through the in vitro cell inhibition assays, we found that Ad-VT has a significant inhibitory effect on ovarian cancer A2780 cells, but no significant inhibitory effect on normal ovarian epithelial cells. Then in vivo experiments showed that Ad-VT significantly inhibited tumor growth and extended the survival time of mice. Subsequent detection of the level of apoptosis found that Ad-VT can cause a strong apoptotic response and kill cells mainly through the endogenous apoptotic pathway. Through the staining analysis of LC3 and the analysis of autophagy-related proteins, it was found that Ad-VT could significantly increase the level of autophagy in A2780 cells, and this was a protective mechanism. CONCLUSIONS: Ad-VT, which replicates under the control of the hTERT promoter and expresses apoptin protein, have significant inhibitory effect on ovarian cancer A2780 cells and promote their apoptosis and autophagy.

Mechanistic insights into acetamiprid-induced genotoxicity on the myocardium and potential ameliorative role of resveratrol.

The current study aimed to explore the genotoxic impacts of the insecticide acetamiprid (ACP) on the myocardium and assess the ameliorative role of resveratrol (RSV). Male rats (10/group) were treated via oral route for 90 days: control; ACP (25 mg/kg); RSV (20 mg/kg); ACP+RSV. Peripheral blood micronucleus test, oxidative stress analysis, comet assay, 8-hydroxydeoxyguanosine and gene expression assessment were performed. The findings revealed that ACP has myocardial genotoxic effects, as demonstrated by increased micronucleus and 8-hydroxydeoxyguanosine formation and increased all comet parameters. Oxidative stress analysis demonstrated that ACP elevated H2O2 and NO levels while decreasing catalase and GST activities. Acetamiprid dysregulated the expression of genes related to oxidative stress and DNA damage response. However, RSV co-treatment resulted in significant protection against these genotoxic impacts. Resveratrol reduced DNA damage and restored the oxidative balance in the myocardium. Moreover, RSV modulated the Nrf2/HO-1 and Atm/P53 pathways, potentiating antioxidant defense and DNA repair.

Dual PI3K/mTOR Inhibitor BEZ235 combined with BMS-1166 Promoting Apoptosis in Colorectal Cancer.

Background: BMS-1166, a PD-1/PD-L1 inhibitor, inhibits the binding of PD-L1 to PD-1, restores T cell function, and enhances tumor immune response. However, mutations in the tumor suppressor or impaired cellular signaling pathways may also lead to cellular transformation. In this study, the SW480 and SW480R cell lines were used as the model to elucidate the treatment with BMS-1166, BEZ235, and their combination. Methods: MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis were analyzed by flow cytometry. The phosphorylation level of the key kinases in the PI3K/Akt/mTOR and MAPK pathways, PD-L1, and the protein levels related to the proliferation, migration, and apoptosis were assessed using western blotting. Results: BEZ235 enhanced BMS-1166-mediated cell proliferation and migration inhibition in SW480 and SW480R cells and promoted apoptosis. Interestingly, the downregulation of the negative regulator PTEN raised the PD-L1 level, which was abolished by the inhibition of Akt. BMS-1166 promoted PI3K, Akt, mTOR, and Erk phosphorylation. However, the combination of BEZ235 with BMS-1166 suppressed the expression of PI3K, p-Akt, p-mTOR, and p-Erk in SW480 and SW480R cells compared to BMS-1166 or BEZ235 single treatment by inhibiting the binding of PD1 to PD-L1. Conclusions: PD-1 binds to PD-L1 and activates the PI3K/mTOR and MAPK pathways, which might be the molecular mechanism of acquired resistance of CRC to BMS-1166. The combination of the two drugs inhibited the phosphorylation of PI3K, Akt, and Erk in the PI3K/mTOR and MAPK pathway, i.e., BEZ235 enhanced the BMS-1166 treatment effect by blocking the PI3K/mTOR pathway and interfering with the crosstalk of the MAPK pathway. Therefore, these findings provide a theoretical basis for BMS-1166 combined with BEZ235 in the trial treatment of colorectal cancer.

Signal-On Detection of Caspase-3 with Methylene Blue-Loaded Metal-Organic Frameworks as Signal Reporters.

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal-organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.

Exotoxins secreted by Clostridium septicum induce macrophage death: Implications for bacterial immune evasion mechanisms at infection sites.

The induction of macrophage death is considered a potential mechanism by which components secreted by Clostridium septicum are used to evade the innate immune response and cause tissue damage. This study aimed to determine the effects of partially purified fractions of extracellular proteins secreted by C. septicum on the death of mouse peritoneal macrophages. Elicited mouse peritoneal macrophages were incubated with partially purified fractions of proteins secreted by C. septicum into the culture medium. After incubation, the protein fraction with a molecular weight ≥100 kDa caused significant cell death in macrophages, altered cell morphology, increased the expression of markers of apoptosis and autophagy, and increased the expression (protein and mRNA) of IL-10 and TNFα. Our data suggest that the proteins secreted by C. septicum (MW, ≥100 kDa) induce cell death in macrophages by promoting autophagy-triggered apoptosis. This study may contribute to our understanding of the molecular mechanism of immune evasion by C. septicum at the infection site.

Polycyclic polyprenylated acylphloroglucinols from the pericarps of Garcinia multiflora champ. ex Benth. with cytotoxic property.

The phytochemical investigation on the pericarps of Garcinia multiflora resulted in the isolation of 12 previously undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs, 1-12) with a variety of skeletons. Their structures were determined by comprehensive spectroscopic analyses, ECD calculations, and single-crystal X-ray diffraction. Compounds 6-9 possess a rare bicyclo[4.3.1]decane skeleton. Additionally, the anti-tumor activity of the 12 isolates was evaluated. The results indicated that compounds 5, 9, and 12 exhibited significant cytotoxicity in a wide range of cancer cell lines, including the human breast cancer MDA-MB-231 cells, human lung cancer A549 cells, human colon cancer SW480 cells and human ovarian cancer HEY cells. Further studies indicated that compound 5 induced cell cycle arrest and apoptosis, to inhibit the growth of MDA-MB-231 cells. Taken together, these findings expand the chemical diversity of PPAPs and further demonstrate the potential of PPAPs as candidates for cancer treatment.

Unveiling the mechanism of hesperidin-induced LdTopI-mediated cell death pathway in protozoan parasite Leishmania donovani.

Unicellular protozoan parasite Leishmania donovani is the causative agent for visceral leishmaniasis (VL) or Kala-azar, a neglected fatal parasitic disease. The conventional treatment of VL consists of therapeutic agents having several shortcomings such as toxicity, high cost, efficacy variance and increased drug resistance. Therefore, there is a desperate need to develop an effective treatment against the parasite. Previous reports suggested that flavonoids can inhibit the enzyme Leishmania donovani DNA topoisomerase I (LdTopILS). Therefore, for the first time in this present study, we divulge HSP (one of the natural sources of flavonoids), as a potent natural antileishmanial compound with efficacy in BALB/c mice at 20 mg/kg of body weight, inhibits LdTopILS at 97 % of its activity at 160 µM in preincubation condition (competitively). It binds with free enzyme and does not allow it to bind with the substrate DNA. Moreover, HSP does not stabilize DNA-topoisomerase I cleavable complex. Thus, HSP acts a catalytic topoisomerase I inhibitor, which inhibits complete activity by binding with Lys269 and Thr411 of large subunit of the enzyme. On the other hand, HSP induces the topo I-mediated programmed cell death process by the formation of cellular reactive oxygen species, resulting in depolarization of mitochondrial membrane potential, followed by fragmentation of nuclear DNA. Therefore, the present study illuminates a natural flavonoid that in future might be a promising lead for the treatment of VL.

Guidance on the assessment of biocompatibility of biomaterials: Fundamentals and testing considerations.

BACKGROUND: Assessing the biocompatibility of materials is crucial for ensuring the safety and well-being of patients by preventing undesirable, toxic, immune, or allergic reactions, and ensuring that materials remain functional over time without triggering adverse reactions. To ensure a comprehensive assessment, planning tests that carefully consider the intended application and potential exposure scenarios for selecting relevant assays, cell types, and testing parameters is essential. Moreover, characterizing the composition and properties of biomaterials allows for a more accurate understanding of test outcomes and the identification of factors contributing to cytotoxicity. Precise reporting of methodology and results facilitates research reproducibility and understanding of the findings by the scientific community, regulatory agencies, healthcare providers, and the general public. AIMS: This article aims to provide an overview of the key concepts associated with evaluating the biocompatibility of biomaterials while also offering practical guidance on cellular principles, testing methodologies, and biological assays that can support in the planning, execution, and reporting of biocompatibility testing.

Cloning and functionally characterization of TtVtg2-like in horseshoe crab Tachypleus tridentatus: A special focus on ovarian development.

Horseshoe crabs are living fossils. In recent decades, the population of horseshoe crabs, especially the tri-spine horseshoe crab Tachypleus tridentatus, has decreased significantly and was listed as an 'endangered species' under the IUCN Red List in 2019. In order to improve the reproduction of T. tridentatus to facilitate stock enhancement, it is important to understand their ovarian development. In this study, a novel TtVtg2-like gene from T. tridentatus was cloned and functionally characterized. The total legth of TtVtg2-like was 5469 bp, encoding a protein consisting of 1822 amino acid with a pI value of 6.51 and a molecular weight of 208.68 KDa. The TtVtg2-like was highly expressed in the ovary and yellow connective tissues, mainly localized in cytoplasm and endoplasmic reticulum vesicles of oocytes and yellow connective tissues, respectively. RNA interference of TtVtg2-like caused the accumulation of ROS, DNA damage, and apoptosis of ovarian primary cells. The results of this study provide useful baseline information for future studies on ovarian development in horseshoe crabs.

Mfn2R364W, Mfn2G176S, and Mfn2H165R mutations drive Charcot-Marie-Tooth type 2A disease by inducing apoptosis and mitochondrial oxidative phosphorylation damage.

Charcot-Marie-Tooth type 2A (CMT2A) is a single-gene motor sensory neuropathy caused by Mfn2 mutation. It is generally believed that CMT2A involves mitochondrial fusion disruption. However, how Mfn2 mutation mediates the mitochondrial membrane fusion loss and its further pathogenic mechanisms remain unclear. Here, in vivo and in vitro mouse models harboring the Mfn2R364W, Mfn2G176S and Mfn2H165R mutations were constructed. Mitochondrial membrane fusion and fission proteins analysis showed that Mfn2R364W, Mfn2G176S, and Mfn2H165R/+ mutations maintain the expression of Mfn2, but promote Drp1 upregulation and Opa1 hydrolytic cleavage. In Mfn2H165R/H165R mutation, Mfn2, Drp1, and Opa1 all play a role in inducing mitochondrial fragmentation, and the mitochondrial aggregation is affected by Mfn2 loss. Further research into the pathogenesis of CMT2A showed these three mutations all induce mitochondria-mediated apoptosis, and mitochondrial oxidative phosphorylation damage. Overall, loss of overall fusion activity affects mitochondrial DNA (mtDNA) stability and causes mitochondrial loss and dysfunction, ultimately leading to CMT2A disease. Interestingly, the differences in the pathogenesis of CMT2A between Mfn2R364W, Mfn2G176S, Mfn2H165R/+ and Mfn2H165R/H165R mutations, including the distribution of Mfn2 and mitochondria, the expression of mitochondrial outer membrane-associated proteins (Bax, VDAC1 and AIF), and the enzyme activity of mitochondrial complex I, are related to the expression of Mfn2.

Synergistic antitumor activity of baicalein combined with almonertinib in almonertinib-resistant non-small cell lung cancer cells through the reactive oxygen species-mediated PI3K/Akt pathway.

Introduction: Almonertinib is an important third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) exhibiting high selectivity to EGFR-sensitizing and T790M-resistant mutations. Almonertinib resistance is a major obstacle in clinical use. Baicalein possesses antitumor properties, but its mechanism of antitumor action against almonertinib-resistant non-small cell lung cancer (NSCLC) remains unelucidated. Methods: CCK-8 assay was used to examine the survival rate of H1975/AR and HCC827/AR cells following treatment for 24 h with different concentrations of baicalein, almonertinib or their combination. The changes in colony formation ability, apoptosis, and intracellular reactive oxygen species (ROS) levels of the treated cells were analyzed using colony formation assay and flow cytometry. Western blotting was performed to detect the changes in protein expressions in the cells. The effects of pre-treatment with NAC on proliferation, apoptosis, and PI3K/Akt signaling pathway were observed in baicalein- and/or almonertinib-treated cells. A nude mouse model bearing subcutaneous HCC827/AR cell xenograft were treated with baicalein (20 mg/kg) or almonertinib (15 mg/kg), and the tumor volume and body mass changes was measured. Results: Both baicalein and almonertinib represses the viability of HCC827/AR and H1975/AR cells in a concentration-dependent manner. Compared with baicalein or almonertinib alone, the combined application of the two drugs dramatically attenuates cell proliferation; triggers apoptosis; causes cleavage of Caspase-3, PARP, and Caspase-9; downregulates the protein expressions of p-PI3K and p-Akt; and significantly inhibits tumor growth in nude mice. Furthermore, baicalein combined with almonertinib results in massive accumulation of reactive oxygen species (ROS) and preincubation with N-acetyl-L-cysteine (ROS remover) prevents proliferation as well as inhibits apoptosis induction, with partial recovery of the decline of p-PI3K and p-Akt. Discussion: The combination of baicalein and almonertinib can improve the antitumor activity in almonertinib-resistant NSCLC through the ROS-mediated PI3K/Akt pathway.

MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia.

Although clinical outcomes in chronic lymphocytic leukemia (CLL) have greatly improved with several approved small molecular inhibitors, acquired resistance does occur, leading to disease progression and eventual death. Thus, the effort to explore novel inhibitors and combination therapeutic regimens is needed. The inhibition of MDM2-p53 interaction to restore p53 function has been regarded as a potential strategy for treating different cancers. We investigated the effects of novel MDM2 inhibitor APG-115 in CLL. We found that APG-115 treatment upregulated the expression of p53, MDM2, and p21 at the mRNA and protein level. APG-115 inhibited cell proliferation, induced apoptosis, and arrested the cell cycle at G0/G1 stage. Moreover, APG-115 inhibited the expression of BCL-2, BCL-xL, and MCL-1, and suppressed the activation of AKT and ERK signaling pathways. APG-115 combined with the BCL2 inhibitor, ABT-199 (venetoclax), led to further inhibition of the expression of BCL-2 family anti-apoptotic proteins and consequently enhanced cell death. Collectively, this study demonstrates that APG-115 activates p53 and thus inhibits multiple pro-survival mechanisms, which provides a rational explanation for APG-115 efficiency in inducing cell apoptosis in CLL. The synergistic effect of APG-115 with ABT-199 suggested a potential combination application in CLL therapy.

Empagliflozin alone and in combination with metformin mitigates diabetes-associated renal complications.

Diabetes mellitus is a major public health concern, often leading to undiagnosed micro- and macrovascular complications, even in patients with controlled blood glucose levels. Recent evidence suggests that empagliflozin and metformin have renoprotective effects in addition to their hypoglycemic action. This study investigated the potential protective effect of empagliflozin and metformin on diabetic renal complications. Forty-two adult male Sprague Dawley rats were randomized into six groups: normal control, diabetic control, metformin (250 mg/kg), empagliflozin (10 mg/kg), and combination therapy groups. Type 2 diabetes was induced in rats by a single intraperitoneal injection of streptozotocin (40 mg/kg) following two weeks of 10% fructose solution in their drinking water. Blood glucose, creatinine, urea nitrogen, inflammatory markers (IL-6, TNF-α), and renal tissue caspase-3 were assessed after eight weeks. Blood glucose, urea, creatinine, serum IL-6, TNF-α, and tissue caspase-3 were significantly decreased in the treatment groups compared to the diabetic group. The histopathological findings revealed that treatment with empagliflozin and/or metformin improved the damage in the renal tissue caused by diabetes-induced nephropathy. Moreover, co-administration of empagliflozin and metformin resulted in even better outcomes. Our data revealed that empagliflozin and metformin could improve renal function and decrease inflammation and apoptosis in diabetic animals, delaying the progression of diabetic nephropathy. Combined treatment with metformin and empagliflozin proved to have an additive protective action on renal tissue.

Allantoin reduces glucotoxicity and lipotoxicity in a type 2 diabetes rat model by modulating the PI3K and MAPK signaling pathways.

Objective: The current study aimed to investigate the potential therapeutic impact of allantoin on diabetes produced by a high-fat diet (HFD) and streptozotocin (STZ) in rats. Subjects and methods: Male Sprague-Dawley rats were fed a high-fat diet to induce insulin resistance, followed by streptozotocin injection to induce diabetes. The effect of oral treatment of allantoin (200, 400 and 800 mg/kg/day) for 8 weeks was evaluated by calculating the alteration in metabolic parameters, biochemical indicators, the oral glucose tolerance tests (OGTT) and hyperinsulinemic-euglycemic clamp tests were performed. Histopathological studies were performed in the liver, kidney and pancreas. Next, the expressions of the MAPK and insulin signaling pathway were measured by Western blot analysis to elucidate the potential mechanism underlying these antidiabetic activities. Results: The administration of allantoin resulted in a significant decrease in fasting blood glucose (FBG) levels, glycogen levels, and glycosylated hemoglobin levels in diabetic rats. Additionally, allantoin therapy led to a dose-dependent increase in body weight growth and serum insulin levels. In addition, the administration of allantoin resulted in a considerable reduction in lipid profile levels and amelioration of histological alterations in rats with diabetes. The administration of allantoin to diabetic rats resulted in a notable decrease in Malondialdehyde (MDA) levels, accompanied by an increase in the activity of antioxidant enzymes in the serum, liver, and kidney. The findings of oral glucose tolerance and hyperinsulinemic-euglycemic clamp tests demonstrated a significant rise in insulin resistance following the administration of allantoin. The upregulation of IRS-2/PI3K/p-Akt/GLUT expression by allantoin suggests a mechanistic relationship between the PI3K/Akt signaling pathway and the antihyperglycemic activity of allantoin. Furthermore, it resulted in a reduction in the levels of TGF-ß1/p38MAPK/Caspase-3 expression in the aforementioned rat tissues affected by diabetes. Conclusions: This study implies that allantoin treats type 2 diabetes by activating PI3K. Additionally, it reduces liver, kidney, and pancreatic apoptosis and inflammation-induced insulin resistance.re.

N-acetylcysteine combined with insulin therapy can reduce myocardial injury induced by type 1 diabetes through the endoplasmic reticulum pathway.

With the development of Type 1 diabetes mellitus (T1DM), various complications can be caused. Hyperglycemia affects the microenvironment of cardiomyocytes, changes endoplasmic reticulum homeostasis, triggers unfolding protein response and eventually promotes myocardial apoptosis. However, insulin therapy alone cannot effectively combat the complications caused by T1DM. Forty adult beagles were randomly divided into five groups: control group, diabetes mellitus group, insulin group, insulin combined with NAC group, and NAC group. 24-hour blood glucose, 120-day blood glucose, 120-day body weight, and serum FMN content were observed, furthermore, hematoxylin-eosin staining, Periodic acid Schiff reagent staining, and Sirius red staining of the myocardium were evaluated. The protein expressions of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase 3, Bcl2, and Bax were detected. Results of the pathological section of myocardial tissue indicated that insulin combined with NAC therapy could improve myocardial pathological injury and glycogen deposition. Additionally, insulin combined with NAC therapy down-regulates the expression of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase3, and Bax. These findings suggest that NAC has a phylactic effect on myocardial injury in beagles with T1DM, and the mechanism may be related to the improvement of endoplasmic reticulum stress-induced apoptosis.

Antitumor effects of IOX1 combined with bevacizumab-induced apoptosis and immunity on colorectal cancer cells.

Colorectal cancer (CRC), as a fatal cancer, is one of the most common cancers worldwide. Although the standard treatment for colorectal cancer is well researched and established, long-term patient survival remains poor, and mortality remains high. Therefore, more and more effective treatment options are needed. To evaluate the efficacy of bevacizumab, the histone demethylase inhibitor IOX1, or their combination for the treatment of colorectal cancer, we examined the effects of IOX1, bevacizumab, and IOX1 combined with bevacizumab on cell activity, proliferation, and migration of colorectal cancer cell lines HCT116, RKO, and CT26 by CCK8, colony formation assay, wound healing assay, and transwell assay. The effects of the drugs alone as well as in combination on apoptosis in colorectal cancer cell lines were examined by flow cytometry and further validated by Western blotting for apoptosis-related proteins. The antitumor effects of treatment alone or in combination on colorectal cancer cells were examined in animal models. Mice were injected subcutaneously with CT26 cells and the growth and immune infiltration in tumor tissues were detected by IHC after drug treatment. We found that IOX1 could effectively inhibit the activity of CRC cells and had a significant inhibitory effect on the proliferation and migration of CRC cells. The apoptosis rate increased in a dose-dependent manner after IOX1 treatment on colorectal cancer cells, and the expression of apoptosis-related proteins changed accordingly. Further combination with bevacizumab revealed that the combination had a more significant effect on the proliferation, migration, and apoptosis of CRC cells than either IOX1 or bevacizumab alone. In vivo experiments have found that both alone and combination drugs can inhibit the growth of mouse tumors, but the effect of combination inhibition is the most obvious. Combination therapy significantly inhibited the expression of proliferative marker (Ki67) in tumor xenograft models, and increased content of antigen-specific CD4+, CD8+T cell growth, and granzymeB (GZMB), which is associated with T cell cytotoxicity, was detected in combination therapy. Immunoassays suppressed the expression of relevant PD-1 and decreased. The anticancer drug bevacizumab and the histone demethylase inhibitor IOX1 may inhibit colon cancer cell growth by regulating apoptosis. The inhibitory effect of combination therapy on tumor growth may be achieved, in part, through upregulation of infiltration-mediated tumor immunity by T lymphocytes. The combination of IOX1 and bevacizumab produced significant synergistic effects. This study aims to provide a new direction for CRC combination therapy.

IL-6/STAT3 signaling pathway induces prostate apoptosis response protein-4(PAR-4) to stimulate malignant behaviors of hepatocellular carcinoma cells.

INTRODUCTION AND OBJECTIVES: Prostate apoptosis response protein-4(PAR-4) is considered a tumor suppressor. However, the role of PAR-4 in hepatocellular carcinoma(HCC) has rarely been reported. The study explores the role of PAR-4 in the malignant behaviors of HCC cells. MATERIALS AND METHODS: TCGA database was applied to analyze the expression of PAR-4 in HCC. Evaluated PAR-4 relationship with clinical parameters and prognosis by tissue microarray; Expression of STAT3, p-STAT3, Src and Ras was detected by Western blotting or laser confocal microscopy. Cell scratch and flow cytometry assays were used to observe IL-6 regulation of the malignant behaviors of HCC cells. The tumorigenic potential of HCC cells in vivo was evaluated in a nude mouse tumor model. RESULTS: Analysis indicated that the expression of PAR-4 in HCC tissues was significantly higher than that in normal liver tissues; and PAR-4 interacted with STAT3. KEGG analysis showed that PAR-4 plays a role in the Janus kinase(JAK)/STAT signaling pathway. The positive expression rate of PAR-4 in HCC tissues was significantly higher than that in adjacent tissues. Positive correlation between IL-6 and PAR-4 expression in the HCC tissues. Exogenous IL-6 significantly promoted the proliferation and migration of HCC cells and up-regulated the expression of PAR-4 and p-STAT3 in HCC cells. Interference of the expression of PAR-4 could reduce the malignant behaviors of HCC cells and inhibit tumorigenesis in a nude mouse tumor model. CONCLUSIONS: PAR-4 expression is positively correlated with HCC; PAR-4 promotes malignant behavior of HCC cells mediated by the IL-6/STAT3 signaling pathway.