Different Degrees of Sulfated Laminaria Polysaccharides Recovered Damaged HK-2 Cells and Inhibited Adhesion of Nano-COM and Nano-COD Crystals.

Purpose: The crystal adhesion caused by the damage of renal tubular epithelial cells (HK-2) is the key to the formation of kidney stones. However, no effective preventive drug has been found. This study aims to explore the recovery effects of four Laminaria polysaccharides (SLPs) with different sulfate (-OSO3-) contents on damaged HK-2 cells and the difference in the adhesion of damaged cells to nanometer calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) before and after recovery. Methods: Sodium oxalate (2.6 mmol/L) was used to damage HK-2 cells to establish a damaged model. SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO3- contents of 0.73%, 15.1%, 22.8%, and 31.3%, respectively, were used to restore the damaged cells, and the effects of SLPs on the adhesion of COM and COD, with a size of about 100 nm before and after recovery, were measured. Results: The following results were observed after SLPs recovered the damaged HK-2 cells: increased cell viability, restored cell morphology, decreased reactive oxygen levels, increased mitochondrial membrane potential, decreased phosphatidylserine eversion ratio, increased cell migration ability, reduced expression of annexin A1, transmembrane protein, and heat shock protein 90 on the cell surface, and reduced adhesion amount of cells to COM and COD. Under the same conditions, the adhesion ability of cells to COD crystals was weaker than that to COM crystals. Conclusions: As the sulfate content in SLPs increases, the ability of SLPs to recover damaged HK-2 cells and inhibit crystal adhesion increases. SLP3 with high -OSO3- content may be a potential drug to prevent kidney stones.

Cell Protection and Crystal Endocytosis Inhibition by Sulfated Laminaria Polysaccharides Against Nano-COM-Induced Oxidative Damage in Renal Epithelial Cells.

Background: The damage to renal tubular epithelial cells is closely related to the formation of kidney stones. At present, research on drugs that can protect cells from damage remains limited. Methods: This study aims to explore the protective effects of four different sulfate groups (-OSO3 -) of Laminaria polysaccharides (SLPs) on human kidney proximal tubular epithelial (HK-2) cells and determine the difference in the endocytosis of nano-sized calcium oxalate monohydrate (COM) crystals before and after protection. COM with a size of 230 ± 80 nm was used to damage HK-2 cells to establish a damage model. The protection capability of SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO3 - contents of 0.73, 15, 23, and 31%, respectively, against COM crystal damage and the effect of SLPs on the endocytosis of COM crystals were studied. Results: Compared with that of the SLP-unprotected COM-injured group, the cell viability of the SLP-protected group was improved, healing capability was enhanced, cell morphology was restored, production of reactive oxygen species was reduced, mitochondrial membrane potential and lysosome integrity were increased, intracellular Ca2+ level and autophagy were decreased, cell mortality was reduced, and internalized COM crystals were lessened. The capability of SLPs to protect cells from damage and inhibit the endocytosis of crystals in cells enhanced with an increase in the -OSO3 - content of SLPs. Conclusions: SLPs with a high -OSO3 - content may become a potential green drug for preventing the formation of kidney stones.

Regulation of Laminaria Polysaccharides with Different Degrees of Sulfation during the Growth of Calcium Oxalate Crystals and their Protective Effects on Renal Epithelial Cells.

The original Laminaria polysaccharide (LP0) was sulfated using the sulfur trioxide-pyridine method, and four sulfated Laminaria polysaccharides (SLPs) were obtained, namely, SLP1, SLP2, SLP3, and SLP4. The sulfated (-OSO3 -) contents were 8.58%, 15.1%, 22.8%, and 31.3%, respectively. The structures of the polysaccharides were characterized using a Fourier transform infrared (FT-IR) spectrometer and nuclear magnetic resonance (NMR) techniques. SLPs showed better antioxidant activity than LP0, increased the concentration of soluble Ca2+ in the solution, reduced the amount of CaOx precipitation and degree of CaOx crystal aggregation, induced COD crystal formation, and protected HK-2 cells from damage caused by nanometer calcium oxalate crystals. These effects can inhibit the formation of CaOx kidney stones. The biological activity of the polysaccharides increased with the content of -OSO3 -, that is, the biological activities of the polysaccharides had the following order: LP0 < SLP1 < SLP2 < SLP3 < SLP4. These results reveal that SLPs with high -OSO3 - contents are potential drugs for effectively inhibiting the formation of CaOx stones.

Antioxidant activity of sulfated Porphyra yezoensis polysaccharides and their regulating effect on calcium oxalate crystal growth.

The nucleation, growth and aggregation of calcium oxalate (CaOx) crystals and the oxidative damage of renal tubular epithelial cells are the key factors to induce kidney stones. In this study, degraded Porphyra yezoensis polysaccharide (PYP0) with 14.14% sulfate group (-OSO3-) content was modified via the sulfur trioxide-pyridine method to obtain three kinds of sulfated P. yezoensis polysaccharides (PYPs), namely, PYPS1, PYPS2, and PYPS3, with -OSO3- group contents of 17.11%, 20.28%, and 27.14% respectively. Fourier transform infrared spectroscopy, 1H NMR, and 13C NMR analyses showed that the -OSO3- groups replaced the hydroxyl groups at the C2, C4, and C6 positions on (1 â†’ 3)-linked ß-D-galactose, the basic structural skeleton unit of PYP0. The antioxidant activity of the PYPSs increased after sulfation, and their scavenging capacity for OH and DPPH free radicals was enhanced with the increase in their -OSO3- group content. Calcium oxalate (CaOx) crystal growth experiments showed that sulfated PYPs promoted the conversion of the thermodynamically stable and sharp CaOx monohydrate (COM) crystals into the thermodynamically unstable and round CaOx dihydrate crystals. With the increase in the -OSO3- group content of the polysaccharides, the concentration of soluble Ca2+ ions in the supernatant increased and the amount of CaOx precipitate decreased. PYPs were nontoxic to human kidney proximal tubular epithelial cells (HK-2) and could protect HK-2 from oxidative damage caused by nano-COM and reduce the level of reactive oxygen species in cells. PYPS3, which had the highest degree of sulfation, had the best protective capability. The results of this work showed that sulfation improved the biological activity of PYPs. This study could provide inspiration for the development of new drugs for the prevention and treatment of kidney stones.

Carboxymethylation of Corn Silk Polysaccharide and Its Inhibition on Adhesion of Nanocalcium Oxalate Crystals to Damaged Renal Epithelial Cells.

The purpose of this study was to explore the repair effect of carboxymethyl-modified corn silk polysaccharide (CSP) on oxidatively damaged renal epithelial cells and the difference in adhesion between cells and calcium oxalate crystals. The CSP was degraded and modified through carboxymethylation. An oxidatively damaged cell model was constructed by oxalate damage to human kidney proximal tubular epithelial (HK-2) cells. Then, the damaged cells were repaired by modified polysaccharides, and the changes in biochemical indexes and adhesion ability between cells and crystals before and after repair were detected. Four modified polysaccharides with carboxyl group (-COOH) contents of 3.92% (CSP0), 7.75% (CCSP1), 12.90% (CCSP2), and 16.38% (CCSP3) were obtained. Compared with CSP0, CCSPs had stronger antioxidant activity, could repair damaged HK-2 cells, and could reduce phosphorylated serine eversion on the cell membrane, the expression of osteopontin (OPN) and Annexin A1, and crystal adhesion. However, its effect on the expression of hyaluronic acid synthase was not substantial. The carboxymethyl modification of the CSP can improve its ability to repair cells and inhibit crystal adhesion and aggregation. A high carboxymethylation degree results in strong polysaccharide activity. CCSPs are expected to reduce the risk of kidney stone formation and recurrence.

The interaction between C/EBPß and TFAM promotes acute kidney injury via regulating NLRP3 inflammasome-mediated pyroptosis.

Sepsis-induced inflammatory damage is a crucial cause of acute kidney injury (AKI), and AKI is an ecumenical fearful complication in approximately half of patients with sepsis. CCAAT/enhancer-binding protein ß (C/EBPß) plays roles in regulating acute phase responses and inflammation. However, the role and mechanism of C/EBPß in AKI are unclear. LPS combined with ATP-treated renal epithelial cells HK2 and cecal ligation-peferation (CLP)-mice were used as models of AKI in vitro and in vivo. Cell damage, the secretion of interleukin-1 beta (IL-1ß), IL-18 and cysteinyl aspartate specific proteinase 1 (caspase-1) activity were tested by LDH, ELISA assay and flow cytometry analysis, respectively. The expression levels of TFAM, C/EBPß, and pyroptosis-related molecules were tested by qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assessed the interaction between C/EBPß with TFAM. Hematoxylin-Eosin (H&E) staining detected pathological changes of kidney tissues, and immunohistochemistry measured TFAM and C/EBPß in mice kidney tissues. C/EBPß or TFAM were up-regulated in LPS combined with ATP -induced HK2 cells. Knockdown of C/EBPß could suppress cell injury and the secretion of IL-1ß and IL-18 induced by LPS combined with ATP. Furthermore, C/EBPß up-regulated the expression levels of TFAM via directly binding to TFAM promoter. Overexpression of TFAM reversed the effects of C/EBPß deficiency on pyroptosis. Knockdown of C/EBPß could inhibit NLRP3 inflammasome-mediated caspase-1 signaling pathway by inactivating TFAM/RAGE pathway. It was further confirmed in the AKI mice that C/EBPß and TFAM promoted AKI by activating NLRP3-mediated pyroptosis. The interaction of between C/EBPß and TFAM facilitated pyroptosis by activating NLRP3/caspase-1 signal axis, thereby promoting the occurrence of AKI.

Upregulation of Mitochondrial Transcription Factor A Promotes the Repairment of Renal Tubular Epithelial Cells in Sepsis by Inhibiting Reactive Oxygen Species-Mediated Toll-Like Receptor 4/p38MAPK Signaling.

BACKGROUND: Mitochondrial transcription factor A (TFAM) plays multiple pathophysiologic roles in mitochondrial DNA (mtDNA) maintenance. However, the role of TFAM in sepsis-induced acute kidney injury (AKI) remains largely unknown. METHODS: Lipopolysaccharide (LPS) treatment of HK-2 cells mimics the in vitro model of AKI inflammation. pcDNA3.1 plasmid was used to construct pcDNA3.1-TFAM. sh-TFAM-543, sh-TFAM-717, sh-TFAM-765, sh-TFAM-904 and pcDNA3.1-TFAM were transfected into HK-2 cells using Lipofectamine 2000. MtDNA transcriptional levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was performed to assess the cell viability. Changes in reactive oxygen species (ROS) and mitochondrial membrane potential in HK-2 cells were detected using the corresponding kits. Immunofluorescence experiment was used to investigate the displacement of TFAM. mRNA and protein expression levels of TFAM and its related genes were measured by qRT-PCR and western blot respectively. Mice in sepsis were administered cecal ligation and puncture surgery. RESULTS: LPS treatment was a non-lethal influencing factor, leading to the upregulation of ROS levels and downregulation of mtDNA copy number and NADH dehydrogenase subunit-1 (ND1) expression, and caused damage to the mitochondria. As the LPS treatment time increased, TFAM was displaced from the periphery of the nucleus to cytoplasm. TFAM reduced ROS and P38MAPK levels by inhibiting toll-like receptor 4 (TLR4) expression, ultimately inhibiting inflammation and repairing mtDNA. CONCLUSIONS: Our results indicate that TFAM repairs mtDNA by blocking the TLR4/ROS/P38MAPK signaling pathway in inflammatory cells, thereby repairing septic tubular epithelial cells, and TFAM may serve as a new target for sepsis therapy.

[Complete Remission Rate and Adverse Ractions of Three Different Chemotherapy Regimens in the Treatment of Adult Patients with Newly Diagnosed Non-M3 AML].

OBJECTIVE: To compare the complete remission rate (CRR) and adverse reaction of the 3 different chemotherapy regimens (daunorubicin, idarubicin, imported idarubicin combined with cytarabine) for the treatment of adult patients with newly diagnosed non-M3 acute myeloid leukemia (AML). METHODS: Seventy-one adult patients with newly diagnosed non-M3 AML were divided into 3 groups: 17 cases treated with daunorubicin plus cytarabine as group A, 24 cases treated with idarubicin plus cytarabine as group B, 30 cases treated with the imported idarubicin plus cytarabine as group C. The curative effects and adverse reactions were compared among the 3 groups after treatment. RESULTS: CCR in group C (86.67%) was significantly higher than that in group A (52.94%) and group B (70.83%), and the CRR in group B was significantly higher than that in group A (P<0.05). The incidence of adverse reaction such as nausea, vomiting, myelosuppression and infection among 3 groups were not statistically significantant (P>0.05). CONCLUSION: The curative effect of idarubicin for the treatment of non-M3 AML patients is better than that of daunorubicin, especially the curative efficiency of imported darubicin is much higher; the adverse reaction after treatment by daunorubicin and idarubicin can be controllable, so daunorubicin and idarubicin can be used as first-line drug for the patients with AML, and patients can choose more appropriate drug according to their own economic ability.

[Role of Non-classical NF-κB Signaling Pathway in B Acute Lymphoblastic Leukemia].

OBJECTIVE: To study the role of non-classical NF-κB signaling pathway in B acute lymphoblastic leukemia (B-ALL). METHODS: The bone marrow samples from 48 patients with B-ALL were collected from March 2015 to March 2016. The real-time quantitative RT-PCR was used for determing mRNA expression levels of NF-κB family members; the NF-κB DNA binding activity in B-ALL cell nucleus was analyzed by ELISA; the apoptosis rate of B-ALL cells alone or co-cultured with bone marrow stromal cells (hBMSC) was determined by flow cytometry. RESULTS: Relative expression level of mRNA for NF-κB family members, including the Rel A, Rel B, P50 and P52 in ALL-B group was statistically significantly higher than that in normal control group (P<0.05). The clinical characteristics of B-ALL patients with different NF-κB activity were not significantly different (P> 0.05); after B-ALL cells cultured alone or co-cultured, the apoptosis rate of Rel A+ / Rel B- group was statistically significantly higher than that in Rel A+ / Rel B+ group (P<0.05); the apoptosis rate of B-ALL cells cultured alone or co-cultured with hBMSC was significantly different. CONCLUSION: Non-classical NF-κB signal marked by Rel B can be used as a new target for B-ALL treatment.