Modified Zhenwu Tang delays chronic renal failure progression by modulating oxidative stress and hypoxic responses in renal proximal tubular epithelial cells.

Background: Tubulointerstitial fibrosis (TIF) is a critical pathological feature of chronic renal failure (CRF), with oxidative stress (OS) and hypoxic responses in renal proximal tubular epithelial cells playing pivotal roles in disease progression. This study explores the effects of Modified Zhenwu Tang (MZWT) on these processes, aiming to uncover its potential mechanisms in slowing CRF progression. Methods: We used adenine (Ade) to induce CRF in rats, which were then treated with benazepril hydrochloride (Lotensin) and MZWT for 8 weeks. Assessments included liver and renal function, electrolytes, blood lipids, renal tissue pathology, OS levels, the hypoxia-inducible factor (HIF) pathway, inflammatory markers, and other relevant indicators. In vitro, human renal cortical proximal tubular epithelial cells were subjected to hypoxia and lipopolysaccharide for 72 h, with concurrent treatment using MZWT, FM19G11, and N-acetyl-l-cysteine. Measurements taken included reactive oxygen species (ROS), HIF pathway activity, inflammatory markers, and other relevant indicators. Results: Ade treatment induced significant disruptions in renal function, blood lipids, electrolytes, and tubulointerstitial architecture, alongside heightened OS, HIF pathway activation, and inflammatory responses in rats. In vivo, MZWT effectively ameliorated proteinuria, renal dysfunction, lipid and electrolyte imbalances, and renal tissue damage; it also suppressed OS, HIF pathway activation, epithelial-mesenchymal transition (EMT) in proximal tubular epithelial cells, and reduced the production of inflammatory cytokines and collagen fibers. In vitro findings demonstrated that MZWT decreased apoptosis, reduced ROS production, curbed OS, HIF pathway activation, and EMT in proximal tubular epithelial cells, and diminished the output of inflammatory cytokines and collagen. Conclusion: OS and hypoxic responses significantly contribute to TIF development. MZWT mitigates these responses in renal proximal tubular epithelial cells, thereby delaying the progression of CRF.

Reticulocyte hemoglobin content associated with the risk of iron deficiency anemia.

Background/Objective: Reticulocyte hemoglobin content (MCHr) was recognized as a rapid and reliable marker for investigating iron deficiency (ID). We hypothesized that MCHr was associated with the risk of iron deficiency anemia in adults. Methods: This is a dual-center case-control study. A total of 806 patients and healthy individuals were recruited from Ruijin Hospital and Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine between January 2021 and December 2021. The participants were categorized into iron deficiency anemia (IDA) group (n = 302), non-IDA group (n = 366), and healthy control group (n = 138). According to the MCHr level, the participants were divided into two groups, i.e. normal MCHr (≥25 pg) and decreased MCHr (<25 pg) group. Multivariate logistic regression analysis and adjusted subgroup analysis were conducted to estimate the relative risk between MCHr and IDA, with confounding factors including age, sex, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), Hematocrit (HCT), serum iron (Fe), ferritin (Ferrit), and total iron binding capacity (TIBC). Results: Compared with the non-IDA, the MCHr level with IDA decreased significantly. ROC curve analysis showed that MCHr had the largest area under the AUC curve. After comprehensive adjustment for confounding factors, individuals with normal level of MCHr exhibited a decreased risk of IDA (OR = 0.68 [0.60, 0.77], P < 0.01), while the risk of IDA was up to 5 times higher for those with decreased MCHr. Conclusion: Our findings supported the hypothesis that MCHr was associated with the risk of IDA in adults and could serve as an indicator of IDA severity. MCHr holds clinical value as an auxiliary diagnostic indicator, providing valuable insights into whether invasive examinations are warranted in the assessment of IDA.

Reversal of the Apoptotic Resistance of Non-Small-Cell Lung Carcinoma towards TRAIL by Natural Product Toosendanin.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively triggers cancer cell death via its association with death receptors on the cell membrane, but exerts negligible side effects on normal cells. However, some non-small-cell lung carcinoma (NSCLC) patients exhibited resistance to TRAIL treatment in clinical trials, and the mechanism varies. In this study, we described for the first time that toosendanin (TSN), a triterpenoid derivative used in Chinese medicine for pain management, could significantly sensitize human primary NSCLC cells or NSCLC cell lines to TRAIL-mediated apoptosis both in vitro and in vivo, while showing low toxicity against human primary cells or tissues. The underlying apoptotic mechanisms involved upregulation of death receptor 5 (DR5) and CCAAT/enhancer binding protein homologous protein, which is related to the endoplasmic reticulum stress response, and is further associated with reactive oxygen species generation and Ca2+ accumulation. Surprisingly, TSN also induced autophagy in NSCLC cells, which recruited membrane DR5, and subsequently antagonized the apoptosis-sensitizing effect of TSN. Taken together, TSN can be used to sensitize tumors and the combination of TRAIL and TSN may represent a useful strategy for NSCLC therapy; moreover, autophagy serves as an important drug resistance mechanism for TSN.

Persistency of Enlarged Autolysosomes Underscores Nanoparticle-Induced Autophagy in Hepatocytes.

The diverse biological effects of nanomaterials form the basis for their applications in biomedicine but also cause safety issues. Induction of autophagy is a cellular response after nanoparticles exposure. It may be beneficial in some circumstances, yet autophagy-mediated toxicity raises an alarming concern. Previously, it has been reported that upconversion nanoparticles (UCNs) elicit liver damage, with autophagy contributing most of this toxicity. However, the detailed mechanism is unclear. This study reveals persistent presence of enlarged autolysosomes in hepatocytes after exposure to UCNs and SiO2 nanoparticles both in vitro and in vivo. This phenomenon is due to anomaly in the autophagy termination process named autophagic lysosome reformation (ALR). Phosphatidylinositol 4-phosphate (PI(4)P) relocates onto autolysosome membrane, which is a key event of ALR. PI(4)P is then converted into phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) by phosphatidylinositol-4-phosphate 5-kinase. Clathrin is subsequently recruited by PI(4,5)P2 and leads to tubule budding of ALR. Yet it is observed that PI(4)P cannot be converted in nanoparticle-treated hepatocytes cells. Exogenous supplement of PI(4,5)P2 suppresses the enlarged autolysosomes in vitro. Abolishment of these enlarged autolysosomes by autophagy inhibitor relieves the hepatotoxicity of UCNs in vivo. The results provide evidence for disrupted ALR in nanoparticle-treated hepatocytes, suggesting that the termination of nanoparticle-induced autophagy is of equal importance as the initiation.

Differential ERK activation during autophagy induced by europium hydroxide nanorods and trehalose: Maximum clearance of huntingtin aggregates through combined treatment.

Accelerating the clearance of intracellular protein aggregates through elevation of autophagy represents a viable approach for the treatment of neurodegenerative diseases. In our earlier report, we have demonstrated the enhanced degradation of mutant huntingtin protein aggregates through autophagy process induced by europium hydroxide nanorods [EHNs: Eu(III)(OH)3], but the underlying molecular mechanism of EHNs mediated autophagy was unclear. The present report reveals that EHNs induced autophagy does not follow the classical AKT-mTOR and AMPK signaling pathways. The inhibition of ERK1/2 phosphorylation using the specific MEK inhibitor U0126 partially abrogates the autophagy as well as the clearance of mutant huntingtin protein aggregates mediated by EHNs suggesting that nanorods stimulate the activation of MEK/ERK1/2 signaling pathway during autophagy process. In contrast, another mTOR-independent autophagy inducer trehalose has been found to induce autophagy without activating ERK1/2 signaling pathway. Interestingly, the combined treatment of EHNs and trehalose leads to more degradation of mutant huntingtin protein aggregates than that obtained with single treatment of either nanorods or trehalose. Our results demonstrate the rational that further enhanced clearance of intracellular protein aggregates, needed for diverse neurodegenerative diseases, may be achieved through the combined treatment of two or more autophagy inducers, which stimulate autophagy through different signaling pathways.

Enhanced transdermal delivery of epidermal growth factor facilitated by dual peptide chaperone motifs.

TD1, a peptide chaperone consisting of the sequence ACSSSPHKHCG, has been shown to facilitate transdermal delivery for protein molecules via either co-administration or the fusion approach. We previously reported that a single TD1 motif, fused to the N-terminus of human epidermal growth factor (hEGF) can significantly enhance the transdermal efficiency of the recombinant EGF protein. In an effort to further increase the transdermal efficiency, we have created EGF fusion proteins harboring dual TD1 motifs: TD1-hEGF-TD1, containing one TD1 motif at both the N- and the Cterminus, and TD1-TD1-hEGF, containing two tandem TD1 motifs at the N-terminus. Both TD1-hEGF-TD1 and TD1- TD1-hEGF proteins, expressed in Escherichia coli and purified to apparent homogeneity, exhibited biological activity similar to unmodified hEGF, as revealed by their relative abilities to stimulate fibroblast growth, promote fibroblast migration, and activate the MAP kinase signaling cascade. On the other hand, both TD1-hEGF-TD1 and TD1-TD1-hEGF proteins exhibited a transdermal efficiency enhancement. The improvement was >5-fold compared to unmodified hEGF and 3-fold over the hEGF fusion protein with only one TD1 motif attached. These findings provided proof-of-concept for improving transdermal delivery of protein actives through rational protein design.

Identification of compound heterozygous mutations in the ITGA2B gene in a Chinese patient with Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by the tendency to hemorrhage and the inability of platelets to aggregate in response to agonists. GT is caused by a defect of the platelet glycoprotein IIb/IIIa complex. The objective of this study was to describe the clinical features and the genetic cause of GT in a 6-year-old girl from south China. METHODS: A three-generation family was studied. The proband patient aged 6 years and her parents undertook examinations of platelet counts, blood film, bleeding time, platelet aggregation, and flow cytometry. All coding exons of the ITGA2B and ITGB3 genes were amplified by polymerase chain reaction (PCR), and direct sequencing was performed for mutational screening on the patient and normal controls consisted of 52 healthy blood donors. Reverse transcription PCR was conducted to test for exon skipping. RESULTS: The proposita patient showed dispersing platelets, prolonged bleeding time, and severely reduced platelet aggregation in response to the physiological agonists adenosine diphosphate (ADP), epinephrine, collagen, and ristocetin. Flow cytometric measurements showed that the contents of alphaIIb and beta3 were significantly decreased. Sequencing results demonstrated two different types of heterozygous mutations existed in the alphaIIb gene (c.2930delG and IVS15-1delG). The compound mutations were also confirmed in the patient's mother and father separately. CONCLUSIONS: The alphaIIbbeta3 deficiency of the proband was caused by two compound ITGA2B mutations, which were first reported in Chinese GT patients. The IVS15-1delG was first confirmed to cause an exon skipping.

[Molecular mechanisms of Glanzmann thrombasthenia caused by alphaII b P126H mutation: an in vitro experiment].

OBJECTIVE: To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha IIb P126H mutation. METHODS: Eukaryotic vector of alpha IIb P126H was constructed by PCR site-directed mutagenesis and then co-transfected with eukaryotic vector PCDM8 II a expressing the subunit beta3 into human renal epithelial cells of the line 293& and Chinese hamster ovarian cancer cells of the line CHO after sequencing. The membrane expression of alpha IIb P126H mutant was analyzed by flow cytometry and the whole expression was confirmed by Western blotting. The alpha IIb P126H mutant subcellular localization was determined by laser confocal scanning microscopy. RESULTS: The 293T cells cotransfected with cDNAs of mutated alpha IIb and wild-type beta3 failed to express alpha II bbeta3 on the cell surface as shown by FACS. Western blotting of the cell lysate showed no detectable mature alpha IIb. Immunofluorescence studies demonstrated proa II bbeta3 complex colocalized with an endoplasmic reticulum (ER) marker, but showed minimal colocalization with an Golgi marker. CONCLUSION: The P126H mutation in alpha IIb prevents transport of the pro-alpha II bbeta3 complex from ER to the Golgi body, thus hindering its maturation and surface expression. The impaired alpha II bp33 transport is responsible for the thrombasthenia.

[Antithrombin deficiency due to heterozygous antithrombin gene mutation and a pedigree study].

OBJECTIVE: To identify the antithrombin (AT) phenotype and gene mutation of a kindred with hereditary antithrombin deficiency. METHODS: Plasma AT activity and AT antigen level of the propositus and his kindred members were determined with chromogenic substrate method and immunoassay, respectively. All the seven exons and intron-exon boundaries of antithrombin gene were analyzed by PCR and direct sequencing of amplified PCR products from the propositus. RESULTS: The propositus AT antigen level was normal but his AT activity was only 65% of normal value suggesting that he had type II AT deficiency. A heterozygous G13830A mutation in exon 6 resulting in Arg393His missense mutation in his AT polypeptide was identified in the propositus. The same phenotype and gene mutation were found in other 3 kindred members. CONCLUSION: The type II AT deficiency found in this kindred is caused by heterozygous G13830A mutation in AT gene.