Validation of hyperthermia as an enhancer of chemotherapeutic efficacy: insights from a bladder cancer organoid model.

OBJECTIVE: This study aimed to evaluate the combined efficacy of hyperthermia and chemotherapy using a bladder cancer organoid model and to explore hyperthermia-related molecular pathways. METHOD: Tumor organoids were generated by embedding RT4 bladder cancer cells into Matrigel. The resulting organoids were treated with pirarubicin or gemcitabine at 37 °C or 42 °C. Proliferation was determined by Ki67 immunofluorescence staining, and apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) assay. RNA sequencing was used to identify the differentially expressed genes. RESULTS: Bladder cancer organoids were successfully established and exhibited robust proliferative abilities. Treatment with gemcitabine or pirarubicin under hyperthermic conditions caused pronounced structural damage to the organoids and increased cell death compared to that in the normothermically treated group. Furthermore, Ki67 labeling and TUNEL assays showed that the hyperthermia chemotherapy group showed a significantly reduced proliferation rate and high level of apoptosis. Finally, RNA sequencing revealed the IFN-γ signaling pathway to be associated with hyperthermia. CONCLUSION: Overall, hyperthermia combined with chemotherapy exerted better therapeutic effects than those of normothermic chemotherapy in grade 1-2 non-muscle-invasive bladder cancer, potentially through activation of the IFN-γ-JAK-STAT pathway.

A key mutation in magnesium chelatase I subunit leads to a chlorophyll-deficient mutant of tea (Camellia sinensis).

Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.

The potential effects of chlorophyll-deficient mutation and tree_age on the accumulation of amino acid components in tea plants.

Albino tea has been receiving growing attention on the tea market due to its attractive appearance and fresh taste, mainly caused by high amino acid contents. Here, variations in the contents of five free amino acids in relation to pigment contents and tree age in two hybrid populations'Longjin 43'(♀) × 'Baijiguan'(♂) and 'Longjin 43'(♀) ×'Huangjinya'(♂) with 334 first filial generation individuals including chlorophyll-deficient and normal tea plants were investigated. The data showed that the contents of main amino acids in all filial generation gradually decreased as plant age increased. Principal component analysis indicated that the amino acid content of individual plant tended to be stable with the growth of plants. Correlation analysis clarified that several main amino acids were significantly negatively correlated with chlorophyll a, chlorophyll b and carotenoid contents. Our results showed that the accumulation of amino acids in tea plant was closely related to leaf color variation and the tree age during growing period.

Theaflavin protects against oxalate calcium-induced kidney oxidative stress injury via upregulation of SIRT1.

Renal tubular cell injury induced by calcium oxalate (CaOx) is a critical initial stage of kidney stone formation. Theaflavin (TF) has been known for its strong antioxidative capacity; however, the effect and molecular mechanism of TF against oxidative stress and injury caused by CaOx crystal exposure in kidneys remains unknown. To explore the potential function of TF on renal crystal deposition and its underlying mechanisms, experiments were conducted using a CaOx nephrocalcinosis mouse model established by glyoxylate intraperitoneal injection, and HK-2 cells were subjected to calcium oxalate monohydrate (COM) crystals, with or without the treatment of TF. We discovered that TF treatment remarkably protected against CaOx-induced kidney oxidative stress injury and reduced crystal deposition. Additionally, miR-128-3p expression was decreased and negatively correlated with SIRT1 level in mouse CaOx nephrocalcinosis model following TF treatment. Moreover, TF suppressed miR-128-3p expression and further abolished its inhibition on SIRT1 to attenuate oxidative stress in vitro. Mechanistically, TF interacted with miR-128-3p and suppressed its expression. In addition, miR-128-3p inhibited SIRT1 expression by directly binding its 3'-untranslated region (UTR). Furthermore, miR-128-3p activation partially reversed the acceerative effect of TF on SIRT1 expression. Taken together, TF exhibits a strong nephroprotective ability to suppress CaOx-induced kidney damage through the recovery of the antioxidant defense system regulated by miR-128-3p/SIRT1 axis. These findings provide novel insights for the prevention and treatment of renal calculus.

Ethanol treatment improves the sensory quality of cherry tomatoes stored at room temperature.

The effects of ethanol treatment on quality characteristics of cherry tomatoes were investigated over 11 days of storage at room temperature (25 °C). Results showed that sensory quality was improved after ethanol treatment, with redder, softer fruits at the edible stage (11 days) compared with control fruit. In addition, the contents of ascorbic acid, sucrose and fructose were elevated after ethanol treatment as well as the concentration of 6-methyl-5-hepten-2-one. Conversely, decreased levels of methyl salicylate (MeSA), guaiacol, (Z)-3-hexenal and (E)-2-hexenal were observed. Selected consumers showed a preference for ethanol-treated cherry tomato fruits compared with controls. Taken together, 0.1% ethanol application has the potential to improve the quality characteristics of cherry tomatoes stored at room temperature.

Ascorbic Acid Supplements and Kidney Stones Incidence Among Men and Women: A systematic review and meta-analysis.

PURPOSE: The relationship of ascorbic acid (AA) supplements and risk of kidney stones among men and women is controversial. This systematic evaluation was performed to obtain comprehensive evidence about the relationship of AA supplements and risk of kidney stones among men and women. MATERIAL AND METHODS: A systematic search of Pubmed, the Cochrane Library, Web of Science, Embase was performed to identify studies that exhibited the relationship of AA supplements and risk of kidney stones among men and women were published up to Mar 2017. Outcomes of interest included kidney stones incidence and risk factors. RESULTS: Four studies estimating the association between AA supplements and risk of kidney stones were included for meta-analysis. The kidney stones incidence was significantly higher in men than women with AA supplements (OR= 1.62; 95% CI: 1.09 to 2.42; P=0.02). AA supplements (250-499mg/d, 1000-1499mg/d) was remarkably correlated with the risk of renal stones among men (OR= 1.14, 95% CI: 1.00 to 1.28, P=0.04; OR= 1.12, 95% CI: 1.11 to 1.13, P<0.00001; respectively). However, AA supplements (500-999 mg/d, >1500 mg/d) did not correlate with the risk of renal stones among men (OR= 1.20, 95% CI: 0.99 to 1.46, P=0.06; OR= 1.28, 95% CI: 1.00 to 1.63, P= 0.05; respectively). In addition, AA supplements (250-499mg/d, 500-999mg/d, 1000-1499mg/d, >1500mg/d) did not remarkably correlate with the risk of renal stones among women (OR= 1.00, 95% CI: 0.82 to 1.22, P=0.98; OR= 1.08, 95% CI: 0.99 to 1.18, P=0.09; OR= 0.99, 95% CI: 0.90 to 1.08, P=0.77; OR= 0.99, 95% CI: 0.99 to 1.09, P=0.88; respectively). CONCLUSIONS: AA supplements was remarkably correlated with higher risk for kidney stones incidence in men, but not in women. Further multicenter, prospective and long-term follow-up RCTs are required to verify these findings.

Effects of postharvest methyl jasmonate treatment on main health-promoting components and volatile organic compounds in cherry tomato fruits.

Effects of postharvest methyl jasmonate (MeJA) treatment on the contents of ascorbic acid and carotenoids, as well as the compositions and contents of volatile organic compounds (VOCs) in cherry tomato fruits were investigated during 11 days of storage at room temperature (25 °C). The results showed that MeJA treatment significantly increased the contents of ascorbic acid and carotenoids, especially lycopene in postharvest cherry tomato fruits. Moreover, MeJA treatment improved the contents of carotenoids derived VOCs such as 6-methyl-5-hepten-2-one (MHO), while had no effect on firmness, sugars and titratable acidity. All above results suggested that the exogenous MeJA application is potential in enhancement of main health-promoting components and VOCs in postharvest cherry tomato fruits.

Discovery of potent and selective acetylcholinesterase (AChE) inhibitors: acacetin 7-O-methyl ether Mannich base derivatives synthesised from easy access natural product naringin.

Naringin, as a component universal existing in the peel of some fruits or medicinal plants, was usually selected as the material to synthesise bioactive derivates since it was easy to gain with low cost. In present investigation, eight new acacetin-7-O-methyl ether Mannich base derivatives (1-8) were synthesised from naringin. The bioactivity evaluation revealed that most of them exhibited moderate or potent acetylcholinesterase (AChE) inhibitory activity. Among them, compound 7 (IC50 for AChE = 0.82 ± 0.08 µmol•L-1, IC50 for BuChE = 46.30 ± 3.26 µmol•L-1) showed a potent activity and high selectivity compared with the positive control Rivastigmine (IC50 for AChE = 10.54 ± 0.86 µmol•L-1, IC50 for BuChE = 0.26 ± 0.08 µmol•L-1). The kinetic study suggested that compound 7 bind to AChE with mix-type inhibitory profile. Molecular docking study revealed that compound 7 could combine both catalytic active site (CAS) and peripheral active site (PAS) of AChE with four points (Trp84, Trp279, Tyr70 and Phe330), while it could bind with BuChE via only His 20.

Total triterpene acids, isolated from Corni Fructus, ameliorate progression of renal damage in streptozotocin-induced diabetic rats.

OBJECTIVE: To investigate whether total triterpene acids (TTAs), isolated from Cornus Fructus, attenuates renal function by reducing oxidative stress and down-regulating the expression of transforming growth factor ß1 (TGF-ß1). METHODS: Diabetes was induced by an injection of streptozotocin (40 mg/kg intravenously). Thirty rats were randomly divided into three groups: control group, diabetic model group and TTAs treatment group (50 mg/kg, intragastrically) administrated for 8 weeks from 5th to 12th week. All rats were anaesthetized and then were killed to remove kidneys. The renal function and redox enzyme system parameters were tested. Glomerular morphology was observed by a light microscopy. Immunohistochemistry and Western blot assays were employed to determine the protein levels of TGF-ß1. RESULTS: TTAs attenuated the levels of urinary protein, serum creatinine and blood urea nitrogen, although it did not significantly reduce the level of glucose. In addition, TTAs decreased the malondialdehyde while increased superoxide dismutase, catalase and glutathione peroxide activities in diabetic rats. The renal pathological changes in TTAs treatment group were ameliorated. Furthermore, TTAs also ameliorated the expression of TGF-ß1. CONCLUSION: TTAs improved renal function via reducing oxidative stress and down-regulation the expression of TGF-ß1 in diabetic rats.

[Protective effect of total flavonoids of herba epimedii on testis degeneration in diabetic mice].

OBJECTIVE: To observe the protective effects and investigate the possible mechanism of total flavonoids of herba epimedii (TFE) on diabetic testopathy in mice. METHODS: Diabetic animal model was produced by a single injection of alloxan ( 70 mg/kg, i.v.) in mice. The mice were randomly divided into 3 groups (n = 10): control group, model group and TFE group (100 mg/kg, p.o.), administrated for 8 weeks continuously. The level of serum testosterone and blood glucose were measured after 24 hours in the last administration. Detect the specific biochemical indicators of testis: superoxide dismutase (SOD), malondialdehyde (MDA). Meanwhile, the morphology of testis was observed under light microscope by HE and MASSION dyeing. Immunohistochemistry was employed to detect the level of matrix metalloprotein (MMP)9. RESULTS: Compared with control group, glucose and the content of MDA in testicular tissues increased while the levels of serum testosterone and SOD decreased remarkably in model group. Detection of pathology showed that the diameters of seminiferous tubules, various grades of spermatogenic cell decreased and collagen fibrosis hyperplasia in testicular tissues, the expression of (MMP9) were decreased in model group. These alterations were significantly improved in TFE group (P < 0.01). CONCLUSION: TFE ameliorated the alterations of testis inalloxan-induced mice through promoting the testosterone release, anti-oxidation and improving the expression of MMP9.

Protective effect of Icariin on the early stage of experimental diabetic nephropathy induced by streptozotocin via modulating transforming growth factor ß1 and type IV collagen expression in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Icariin is a major constituent of flavonoid isolated from the plant Herba epimedii, which is used as a traditional Chinese medicine to nourish the kidney and reinforce yang. Therefore, the pharmacological studied of its diabetic nephropathy effect was undertaken to validate its traditional use. MATERIALS AND METHODS: Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (40 mg/kg i.v.). Sustained blood glucose levels (>16.7 mmol/l) were considered as diabetic and selected for experimentation. Thirty rats were randomly divided into three groups: control, diabetic, diabetic+Icariin (80 mg/kg, i.g.) administered 8 weeks from 5th to 12th week. Experiment was carried out at the beginning of 13th week. All rats were anaesthetized and then were killed to remove kidneys. Blood glucose, serum creatinine (Cr), blood urea nitrogen (BUN), the activity of superoxide dismutase (SOD), malondialdehyde (MDA) and hydroxyproline (Hyp) in the kidney tissue were measured. Glomerular morphology was observed by light microscopy. Immunohistochemistry and Western blot were employed to determine the proteins levels of TGF-ß(1) and type IV collagen. RESULTS: The enhancement of Cr and BUN was found in model group, which was significantly attenuated by treatment with Icariin. Meanwhile, elevated MDA and Hyp levels in renal tissue as well as decreased SOD activities in renal tissue were significantly remitted by Icariin. The renal pathological changes in Icariin treatment group were ameliorated. Furthermore, the Icariin decreased the expression of TGF-ß(1) and collagen IV protein. CONCLUSIONS: Icariin can evidently relieve renal damage in rats with diabetic nephropathy induced by STZ, which might be related to modulating the expression of collagen IV and TGF-ß(1) protein.

Antiproliferative activity of the total saponin of Solanum lyratum Thunb in Hela cells by inducing apoptosis.

Total saponin of Solanum lyratum Thunb (TSSLT), a species of natural biologically active substances isolated from Solanum lyratum Thunb, possesses various bioactivities. It has been proposed that the induction of apoptosis may be the basis of its antitumor activity. However, the molecular mechanism underlying the total saponin-induced apoptotic process remains unknown. In the present study, we describe the anti-proliferative effect of TSSLT on human cervical cancer cells (Hela). The TSSLT induced apoptosis of Hela in a time-dependent manner with an IC50 for cell viability of 6 microg/ml. The TSSLT-induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-like activities, poly (ADP-ribose) polymerase (PARP) cleavage and release of cytochrome c (cyt c) into cytosol. TSSLT activated various caspases such as caspase-3, -8, and -9 (like) activities but not caspase-1 like activity. The cell death was completely prevented by the pan caspase inhibitor benzyloxy carbonyl-Val-Ala-Asp- fluoromethyl-ketone (Z-VAD-FMK). More than 80% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, treatment with TSSLT induced the increase of Bax:Bcl-2 ratio in Hela cells. These results suggest that the induction of apoptosis by TSSLT involves multiple pathways antigen including death receptor and mitochondrial pathway and strongly suggest that the mitochondrial pathway was mediated by low expression of Bcl-2 and upregulation of Bax, release of cyt c and subsequent activation of caspase-3 followed by down stream events leading to apoptotic cell death.

Ethanol extracts of Rehmannia complex (Di Huang) containing no Corni fructus improve early diabetic nephropathy by combining suppression on the ET-ROS axis with modulate hypoglycemic effect in rats.

AIM: Liuwei Dihuang (Rehmannia complex, RC) decoction, a classic prescription of Traditional Chinese Medicine (TCM), has been used in treating diabetic nephropathy (DN). Among the 6 crude medicines which contains Corni fructus is recognized as the active fraction for its effectiveness. We aimed to investigate, first, if without Corni fructus a modified RC could be still effective, second, if the ethanol extracts could be better than that of water extract and third, the beneficial effect is mainly stemmed from suppressing the endothelin (ET-1) pathway associated with a moderate hypoglycemic effect. METHODS AND MATERIALS: Diabetes for 8 weeks was induced by a single dose of streptozotocin (STZ, 65 mg/kg, i.p.) in rats and treated with RC extracts in either 95%, 70% ethanol or water separately during 5-8th week. The efficacy of extracts was compared with aminoguanidine (AMG). RESULTS: An increase in albumin and creatinine in 24h urine, blood urea nitrogen (BUN) was found in STZ rats. Oxidative stress was found in renal cortex in association with upregulated plasma ET-1 and mRNA of ETA, decreased MMP 2,9 (matrix matelloproteinases) and increased hydroxyproline. CONCLUSIONS: The RC without Corni fructus was very effective in alleviating DN and ethanol extracts provided greater effects against water extracts. The efficacy in alleviating DN is attributed to normalizing the activated ET system, oxidative stress and MMP 2,9 in combination with a moderate hypoglycemic activity.