Single-cell transcriptome analysis reveals the association between histone lactylation and cisplatin resistance in bladder cancer.

Patients with bladder cancer (BCa) frequently acquires resistance to platinum-based chemotherapy, particularly cisplatin. This study centered on the mechanism of cisplatin resistance in BCa and highlighted the pivotal role of lactylation in driving this phenomenon. Utilizing single-cell RNA sequencing, we delineated the single-cell landscape of Bca, pinpointing a distinctive subset of BCa cells that exhibit marked resistance to cisplatin with association with glycolysis metabolism. Notably, we observed that H3 lysine 18 lactylation (H3K18la) plays a crucial role in activating the transcription of target genes by enriching in their promoter regions. Targeted inhibition of H3K18la effectively restored cisplatin sensitivity in these cisplatin-resistant epithelial cells. Furthermore, H3K18la-driven key transcription factors YBX1 and YY1 promote cisplatin resistance in BCa. These findings enhance our understanding of the mechanisms underlying cisplatin resistance, offering valuable insights for identifying novel intervention targets to overcome drug resistance in Bca.

Intestinal dysbiosis exacerbates susceptibility to the anti-NMDA receptor encephalitis-like phenotype by changing blood brain barrier permeability and immune homeostasis.

Changes in the intestinal microbiota have been observed in patients with anti-N-methyl-D-aspartate receptor encephalitis (NMDARE). However, whether and how the intestinal microbiota is involved in the pathogenesis of NMDARE susceptibility needs to be demonstrated. Here, we first showed that germ-free (GF) mice that underwent fecal microbiota transplantation (FMT) from NMDARE patients, whose fecal microbiota exhibited low short-chain fatty acid content, decreased abundance of Lachnospiraceae, and increased abundance of Verrucomicrobiota, Akkermansia, Parabacteroides, Oscillospirales, showed significant behavioral deficits. Then, these FMT mice were actively immunized with an amino terminal domain peptide from the GluN1 subunit (GluN1356-385) to mimic the pathogenic process of NMDARE. We found that FMT mice showed an increased susceptibility to an encephalitis-like phenotype characterized by more clinical symptoms, greater pentazole (PTZ)-induced susceptibility to seizures, and higher levels of T2 weighted image (T2WI) hyperintensities following immunization. Furthermore, mice with dysbiotic microbiota had impaired blood-brain barrier integrity and a proinflammatory condition. In NMDARE-microbiota recipient mice, the levels of Evan's blue (EB) dye extravasation increased, ZO-1 and claudin-5 expression decreased, and the levels of proinflammatory cytokines (IL-1, IL-6, IL-17, TNF-α and LPS) increased. Finally, significant brain inflammation, mainly in hippocampal and cortical regions, with modest neuroinflammation, immune cell infiltration, and reduced expression of NMDA receptors were observed in NMDARE microbiota recipient mice following immunization. Overall, our findings demonstrated that intestinal dysbiosis increased NMDARE susceptibility, suggesting a new target for limiting the occurrence of the severe phenotype of NMDARE.

Fluid absorption during flexible ureteroscopy with intelligent control of renal pelvic pressure: a randomized controlled trial.

PURPOSE: To investigate fluid absorption and its influencing factors during flexible ureteroscopy with intelligent control of renal pelvic pressure (RPP). METHODS: A total of 80 patients with upper urinary tract calculi underwent flexible ureteroscopy with intelligent control of RPP by pressure-measuring ureteral access sheath and were randomly divided into four groups. The RPP of Groups A, B, and C were set at - 5, 0 and 5 mmHg, respectively. Conventional flexible ureteroscopy with uncontrolled pressure served as control Group D. The perfusion flow rate was set at 100 ml/min in the four groups, with 20 patients in each group. The fluid absorption was measured by 1% ethanol every 10 min. Operation time, stone-free rate, and complications were recorded. RESULT: Seventy-three patients were finally included in the RCT. The general and preoperative data of the patients were comparable between the groups. The fluid absorption of Groups A, B, and C was significantly less than that of Group D (P < 0.01). Fluid absorption and operation time were positively correlated, and the correlation coefficients R were 0.864, 0.896, 0.918, and 0.947, respectively (P < 0.01). The fluid absorption of patients with vomiting, fever and ureteral injury was greater than that of patients without complications in the four groups (P < 0.01). In different groups, fluid absorption was greater in patients with ureteral injury Post-Ureteroscopic Lesion Scale (PULS) 1-3 than in noninjured patients (P < 0.01). CONCLUSION: Flexible ureteroscopy with intelligent control of RPP effectively reduces the absorption of perfusion fluid. Operation time and ureteral injury are also key factors affecting perfusion fluid absorption. REGISTRATION NUMBER AND DATE: NCT05201599; August 11, 2021.

Flexible Ureteroscopy with a Tip-Flexible Pressure-Controlling Ureteral Access Sheath for Renal Stones in Children.

INTRODUCTION: The aim of this study was to evaluate the safety and efficacy of flexible ureteroscopy using a tip-flexible pressure-controlling ureteral access sheath (TFPC-UAS) for renal stones in children. METHODS: Consecutive patients aged 5-18 years with renal stones of diameter 1-3 cm were enrolled between January 2022 and November 2023 at Ganzhou People's Hospital. The patients were treated with flexible ureteroscopy using the TFPC-UAS. The renal pelvic pressure (RPP) parameters were set as follows: control value at -10 mm Hg to 5 mm Hg, warning value at 20 mm Hg, and limit value at 30 mm Hg. The infusion flow rate was set to 100-120 mL/min. A holmium laser (276 µm) was used to fragment the stone at 2.0-2.5 J/pulse with a frequency of 20-30 pulses/s. The cases were analyzed for RPP, operative time, stone-free rate, and complications. RESULTS: A total of 21 consecutive patients were included. Two patients were switched to percutaneous nephrolithotomy owing to sheath placement failure. The RPP was -4.6 ± 2.1 mm Hg. The mean operative time was 56.5 ± 17.1 min. The postoperative hospitalization time was 1.5 ± 0.3 days. The stone-free rates at 1 day and 1 month after surgery were 81.0% and 85.7%, respectively. Residual stones in 2 patients were cleared after extracorporeal shockwave lithotripsy. Three cases of Clavien I complications and one case of Clavien II complications occurred. No major complications (Clavien grade III-V) were observed. CONCLUSIONS: Flexible ureteroscopy with a TFPC-UAS is safe and effective for renal stones in children.

Ureteroscopic lithotripsy with pressure-measuring ureteral access sheath for large ureteral stones.

INTRODUCTION: To evaluate the safety and efficacy of ureteroscopic lithotripsy with pressure-measuring ureteral access sheath (PM-UAS) for large ureteral stones. MATERIAL AND METHODS: A total of 258 consecutive patients with large ureteral stones ≥15 mm was enrolled. They were treated by ureteroscopic lithotripsy with PM-UAS in the oblique supine lithotomy position. The technology can precisely monitor and automatically control cavity pressure. The cavity pressure control value was set at -15 mmHg∼-5 mmHg. The cavity pressure limit value was set at 30 mmHg. Infusion flow rate was set at 100-200 ml/min. Postoperative data such as stone-free rate and complications were analyzed. RESULTS: PM-UAS was successfully implanted in 225 patients at one stage. Eighteen cases of patients who had failed the first surgery were successfully treated with a second operation. Fifty-one cases with stones migrating up to the kidney were converted to flexible lithotripsy. The other 15 cases were converted to percutaneous nephrolithotomy due to significant ureteral stenosis. The operative time was 49.5 ± 11.2 min. The stone-free rates after one month and three months were 87.2% (212/243) and 94.2% (229/243), respectively. Complications from grade I to II were observed in 25(10.3%) patients. No other complications from grade III to V were noted. CONCLUSION: The ureteroscopic lithotripsy with PM-UAS is safe and efficacious for large ureteral stones.

Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

A mangrove endophytic fungus Phomopsis asparagi DHS-48 was found to be particularly productive with regard to the accumulation of substantial new compounds in our previous study. In order to explore its potential to produce more unobserved secondary metabolites, epigenetic manipulation was used on this fungus to activate cryptic or silent genes by using the histone deacetylase (HDAC) inhibitor sodium butyrate and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-Aza). Based on colony growth, dry biomass, HPLC, and 1H NMR analyses, the fungal chemical diversity profile was significantly changed compared with the control. Two new compounds, named phaseolorin J (1) and phomoparagin D (5), along with three known chromones (2-4) and six known cytochalasins (6-11), were isolated from the culture treated with sodium butyrate. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD and 13C NMR calculations. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated. Compounds 1 and 8 moderately inhibited the proliferation of ConA (concanavalin A)-induced T and LPS (lipopolysaccharide)-induced B murine spleen lymphocytes. Compound 5 exhibited significant in vitro cytotoxicity against the tested human cancer cell lines Hela and HepG2, which was comparative to the positive control adriamycin and fluorouracil. Our finding demonstrated that epigenetic manipulation should be an efficient strategy for the induction of new metabolites from mangrove endophytic fungi.

Suctioning Flexible Ureteroscopy with Automatic Control of Renal Pelvic Pressure versus Mini PCNL for the Treatment of 2-3-cm Kidney Stones in Patients with a Solitary Kidney.

OBJECTIVE: The aim of the study was to compare the treatment outcomes between suctioning flexible ureteroscopic lithotomy (SF-URL) with automatic control of renal pelvic pressure and minimally invasive percutaneous nephrolithotomy (MPCNL) for the management of 2-3-cm renal stones in patients with a solitary kidney. MATERIALS AND METHODS: A total of 127 patients with a solitary kidney who underwent SF-URL (n = 57) or MPCNL (n = 70) for large renal stones (>2 cm) between June 2015 and October 2020 were consecutively analyzed. The stone characteristics, operative times, stone-free rate (SFR), hospital stays, and incidences of complications were compared. RESULTS: There was a significantly shorter operative time with MPCNL than with SF-URL (43.4 ± 18.9 min vs. 61.8 ± 21.1 min, p = 0.012). SFR at 30 days were 80.7% (46/57) and 90.0% (63/70) for SF-URL and MPCNL, respectively (p > 0.05). The SFR at the 3-month follow-up was comparable in both groups (91.2% vs. 95.7%, p > 0.05). The hemoglobin decline value, hospital stay, serum cystatin C, and percentage of patients requiring blood transfusions in the SF-URL group were obviously better than those in the MPCNL group: (0.8 ± 0.4) versus (3.9 ± 2.7) g/dL (p = 0.007), (3.6 ± 1.5) versus (6.9 ± 3.1) days (p = 0.013), (1.02 ± 0.48) versus (2.54 ± 0.69) mg/L (p = 0.011), and 0 (0.0%) versus 7 (10.0%) (p = 0.016), respectively. The percentages of patients with thrombosis and perirenal hematoma in the MPCNL group were higher than those in the SF-URL group, but the difference was not statistically significant (p > 0.05). CONCLUSION: For the treatment of 2-3-cm renal stones in patients with a solitary kidney, both SF-URL and MPCNL are effective. MPCNL has the advantage of a shorter operation time. However, SF-URL is characterized by less bleeding, shorter hospital stay, and less damage to kidney function.

Betulinic acid promotes the osteogenic differentiation of human periodontal ligament stem cells by upregulating EGR1.

Periodontitis is one of the most common chronic inflammations of the oral cavity, which eventually leads to tooth loss. Betulinic acid (BetA) is an organic acid that has anti-inflammatory effects and is derived from fruits and plants, but its effect on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is still unclear. This study aimed to explore the effect of BetA on the osteogenic differentiation of hPDLSCs and its mechanism. Our results revealed that BetA not only promoted the viability of hPDLSCs but also induced their osteogenic differentiation in a dose-dependent manner. In addition, RNA sequencing was used to screen the differentially expressed genes (DEGs) after hPDLSCs were treated with BetA, and 127 upregulated and 138 downregulated genes were identified. Gene Ontology enrichment analysis showed that DEGs were mainly involved in the response to lithium ions and the positive regulation of macrophage-derived foam cell differentiation. The Kyoto Encyclopedia of Genes and Genomes analysis results revealed that DEGs were enriched in the nuclear factor-κB and interleukin-17 signaling pathways. More importantly, we confirmed that early growth response gene 1 (EGR1), one of the three DEGs involved in bone formation, significantly promoted the expression of osteogenic markers and the mineralization of hPDLSCs. Knockdown of EGR1 obviously limited the effect of BetA on the osteogenic differentiation of hPDLSCs. In conclusion, BetA promoted the osteogenic differentiation of hPDLSCs through upregulating EGR1, and BetA might be a promising candidate in the clinical application of periodontal tissue regeneration.

MicroRNA-146a-5p Downregulates the Expression of P-Glycoprotein in Rats with Lithium-Pilocarpine-Induced Status Epilepticus.

Increasing evidence supports that the efflux transporters, especially P-glycoprotein (P-gp), have vital roles on drug resistance in epilepsy. Overexpression of P-gp in the brain could reduce the anti-epileptic drugs (AEDs) concentration in the epileptogenic zone, resulting in drug resistance. Studies have demonstrated that recurrent seizures induce the expression of P-gp and status epilepticus (SE) could upregulate the expression of P-gp, resulting in drug resistance. MicroRNAs (miRNAs), as endogenous regulators, represent small regulatory RNA molecules that have been shown to act as negative regulators of gene expression in different biological processes. We investigated the impact of miR-146a-5p on the expression of P-gp in status epilepticus rat model. The expression of miR-146a-5p in rat cortex and hippocampus was measured by quantitative RT-PCR at 2 weeks after induction of SE. Meanwhile, we detected the expression of P-gp in the brain of SE rats using Western blotting and immunohistochemistry. Upregulation of miR-146a-5p and overexpression of P-gp were evident at 2 weeks after SE. Moreover, the expression of P-gp was downregulated by injection of miR-146a mimic into the hippocampus. We also detected the expression of interleukin-1 receptor-associated protein kinases-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) and nuclear factor-kappaB (NF-κB) p65 using Western blotting and immunohistochemistry, which indicated the expression of IRAK1, TRAF6 and NF-κB p-p65/p65 increased in the brain of SE rats, and overexpression of miR-146a-5p could downregulate the expression of IRAK1, TRAF6, NF-κB p-p65/p65 and P-gp. Our study indicated that miR-146a-5p may decrease the expression of P-gp in status epilepticus rats via NF-κB signaling pathway.

Nanoparticle-microRNA-146a-5p polyplexes ameliorate diabetic peripheral neuropathy by modulating inflammation and apoptosis.

Nontoxic and nonimmunogenic nanoparticles play an increasingly important role in the application of pharmaceutical nanocarriers. The pathogenesis of diabetic peripheral neuropathy (DPN) has been extensively studied. However, the role of microRNAs in DPN remains to be clarified. We verified in vitro that miR-146a-5p mimics inhibited the expression of proinflammatory cytokines and apoptosis. Then, we explored the protective effect of nanoparticle-miRNA-146a-5p polyplexes (nano-miR-146a-5p) on DPN rats. We demonstrated that nano-miR-146a-5p improved nerve conduction velocity and alleviated the morphological damage and demyelination of the sciatic nerve of DPN rats. The expression of the inflammatory cytokines, caspase-3, and cleaved caspase-3 in the sciatic nerve was inhibited by nano-miR-146a-5p. Additionally, nano-miR-146a-5p increased the expression of myelin basic protein. These results all indicated that nano-miR-146a-5p had a protective effect on peripheral nerves in the DPN rat model, which may occur through the regulation of the inflammatory response and apoptosis.

A Novel Technique to Intelligently Monitor and Control Renal Pelvic Pressure during Minimally Invasive Percutaneous Nephrolithotomy.

OBJECTIVE: To introduce a novel technique for intelligently monitoring and controlling renal pelvic pressure (RPP) in minimally invasive percutaneous nephrolithotomy (MPCNL) and to investigate its reliability and stability. MATERIALS AND METHODS: A total of 63 kidney stone patients (41 males and 22 females) were enrolled in the study. The average stone size was 3.7 ± 1.1 cm. The average age was 41.6 ± 15.6 years old. All patients underwent MPCNL under combined spinal and epidural anesthesia in prone position. A ureteral catheter connected to an invasive blood pressure monitor was retrogradely placed to measure renal pelvic outlet pressure. The MPCNL was performed with the aid of the patented device, including an irrigation and suctioning platform and a pressure-measuring suctioning sheath. On the platform, the RPP control value was set at -5 mm Hg, and the RPP warning value was set at 20 mm Hg. RPP was measured during the irrigation and suctioning period (ISP), and therapeutic period (TP) when the infusion flow was set at 300, 400, and 500 mL/min, respectively, for 5 min. RESULTS: Sixty-three patients successfully underwent the procedure without serious complications. The mean operative time was 67 min (range 31-127 min). Three patients had residual stones >2 mm in size. No statistical significance was observed between the renal pelvic outlet pressure, platform RPP values, and RPP control values for the 300, 400, and 500 mL/min groups during the ISP and TP (p > 0.05). CONCLUSION: The patented devices including the platform and the sheath can reliably and stably monitor and control RPP in real time and within a safe range during MPCNL.

Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.

Suctioning Minimally Invasive Percutaneous Nephrolithotomy with a Patented System Is Effective to Treat Renal Staghorn Calculi: A Prospective Multicenter Study.

OBJECTIVES: To investigate the safety, efficacy, and practicability of minimally invasive percutaneous nephrolithotomy (MPCNL) with the aid of a patented irrigation clearance system in treating renal staghorn calculi. METHODS: From August 2009 to July 2014, 4 hospitals had executed a prospective multicenter study with a total of 912 cases. The patients were randomly divided into 3 groups: suctioning MPCNL, standard percutaneous nephrolithotomy (PCNL), and traditional MPCNL groups. Multiple operative and perioperative parameters were compared. RESULTS: Blood loss and intrapelvic pressure in the suctioning MPCNL group were significantly less than those in the standard PCNL group. The average operation time, intrapelvic pressure, and amount of bleeding in the suctioning MPCNL group were better than those in the traditional MPCNL group. The suctioning MPCNL used one tract more frequently and 2 or 3 tracts less frequently than the standard MPCNL and traditional MPCNL groups. The stone-free rate by one surgery in the suctioning MPCNL group was significantly higher than that in standard PCNL and traditional MPNCL groups. CONCLUSIONS: Suctioning MPCNL using our patented system shows several advantages in treating renal staghorn calculi, including minimal invasion, shorter operation time, lower intrapelvic pressure, less bleeding and the need for a smaller number of -percutaneous tracts, and higher stone clearance rate by one -surgery.

A Randomized Study of Minimally Invasive Percutaneous Nephrolithotomy (MPCNL) with the aid of a patented suctioning sheath in the treatment of renal calculus complicated by pyonephrosis by one surgery.

BACKGROUND: Calculus pyonephrosis is difficult to manage. The aim of this study is to explore the value of a patented suctioning sheath assisted minimally invasive percutaneous nephrolithotomy (MPCNL) in the treatment of calculus pyonephrosis. METHODS: One hundred and eighty two patients with calculus pyonephrosis were randomizely divided into observation group (n = 91) and control group (n = 91). The control group was treated with MPCNL traditionally using peel-away sheath while the observation group was treated with MPCNL using the patented suctioning sheath. RESULTS: All the patients in the observation group underwent one stage surgical treatment, 14 patients in the control group underwent first-stage surgery with the rest of the group underwent one stage surgery. The complication rate was 12.1% in the observation group, significantly lower than the rate in the control group which was 51.6%; One surgery stone clearance in the observation group was 96.7% while it was 73.6% in the control group; operative time in the observation group was (54.5 ± 14.5) min, compared to (70.2 ± 11.7) min in the control group; the bleeding amount in the observation group was (126.4 ± 47.2) ml, compared to (321.6 ± 82.5) ml in the control group; the hospitalization duration for the observation group was (6.4 ± 2.3) days, compared to (10.6 ± 3.7) days in the control group. Comparison of the above indicators, the observation group was better than the control group with significant difference (p < 0.001 each). CONCLUSIONS: Minimally invasive percutaneous nephrolithotomy with the aid of the patented suctioning sheath in the treatment of calculus pyonephrosis in one surgery is economic, practical, and warrants clinical promotion. TRIAL REGISTRATION: This study was registered with Chinese Clinical Trial Registry on May 18, 2016 (retrospective registration) with a trial registration number of ChiCTR-IOR-16008490 .

The New Generation Mini-PCNL System - Monitoring and Controlling of Renal Pelvic Pressure by Suctioning Device for Efficient and Safe PCNL in Managing Renal Staghorn Calculi.

INTRODUCTION: In order to better control renal pelvic pressure (RPP), reduce surgical complications and increase operative efficacy of minimally invasive percutaneous nephrolithotomy (MPCNL), we designed an MPCNL with intelligent monitoring and control of RPP and sought to explore its clinical efficacy and characteristics. SUBJECT AND METHODS: This study included 60 patients with renal staghorn calculi. A 5F ureteral catheter was connected to an invasive blood pressure monitor for monitoring and control of RPP. A 12F renoscope was inserted through a percutaneous tract for lithotripsy using a laser, with the help of an intelligent system to monitor RPP continuously, which includes an irrigation and suctioning platform and an integrated intraluminal pressure sensory PCNL suctioning sheath. Before the lithotripsy, intraluminal pressure control value and perfusion flow were set on the platform. RESULTS: RPP was maintained at a range of -12 to 2 mm Hg. The mean operative time was 120 min. The mean intraoperative bleeding amount was 100 ml. Fifty-two of the 60 patients the stones were cleared by one surgery. CONCLUSIONS: In our suctioning MPCNL with the intelligent control of RPP, the intraoperative RPP could be accurately controlled within the preset safety range. The stone clearance rate was high.

Predicting Outcomes after Minimally Percutaneous Nephrolithotomy with the Aid of a Patented System by Using the Guy's Stone Score.

OBJECTIVES: At present, there is no report on the application of Guy's stone (GS) score in minimally invasive percutaneous nephrolithotomy (MPCNL). We therefore investigate the clinical effect of MPCNL by using a patented irrigation and clearance system according to the GS. METHODS: A total of 222 consecutive patients were divided into 2 groups that were treated with MPCNL using the patented system or standard PCNL using Electro Medical System (EMS) ultrasound/ballistic trajectory lithotripsy and clearance system, respectively (124 patients in patented system group, 98 patients in EMS system group). The stone complexity and severity of complications were stratified by GS and Modified Clavien grading system in this study, respectively. The success and complication rates between MPCNL and PCNL were compared. RESULTS: The patented system group had a significantly higher success rate (GS3: 95 vs. 43% and GS4: 67 vs. 31%) and a significantly less complications rate (GS3: 12 vs. 27% and GS4: 21 vs. 46%) in GS3 and GS4 compared with the EMS system group. Also, the patented system had significant lower complication rates in Clavien grades II-IV than that of the EMS system. CONCLUSION: The MPCNL using the patented irrigation and clearance system was more efficient in dealing with renal stones of high complexity (GS3-4).

[Magnetic Resonance Imaging Tracing the Biodistribution of SPIO-shRNA Molecular Probe in vivo].

OBJECTIVES: To investigate the biodistribution of superparamagnetic iron oxide (SPIO)-shRNA molecular probe by magnetic resonance imaging (MRI) in vivo. METHODS: Six New Zealand white rabbits were injected intravenously with SPIO-shRNA molecular probe (9.6 mg Fe/kg) via ear edge vein. The blood samples were collected to analyse the pharmacokinetic parameters through measuring the iron content by atomic absorption spectrometry (AAS) method at 30 min before and 1 min, 3 min, 5 min, 10 min, 15 min, 30 min and at 1, 2, 3, 6, 12, and 24 h after the injection. Six Kun Ming (KM) mice were injected intravenously with SPIO-shRNA molecular probe (4.8 mg Fe/kg). The biodistribution of SPIO-shRNA molecular probe was traced by MRI in vivo. Ninety six KM mice were randomly divided into control group and experimental group: each mouse in experimental group was injected intravenously with SPIO-shRNA molecular probe (4.8 mg Fe/kg). The liver, spleen, kidney, brain and muscle of the control group and the experimental group on 1, 3, 5, 7, 9, 11 and 14 d after the injection were collected. The organ iron content were measured by AAS method and Prussian blue staining in order to observe the distribution of the SPIO-shRNA molecular probe in the main organ. RESULTS: Our results demonstrated that the pharmacokinetics of the molecular probe complied with two-compartment model, and the blood half-life was (3.692±0.196) h. The data of MRI showed that the probe were distributed in liver and spleen, and the signs were reduced in accord with the increase of probe's doses in liver and spleen. The probe's metabolism was slow, and the probe was cleared from liver and spleen at 2 weeks after the injection. The results of AAS and Prussian blue staining further testified the results of MRI. CONCLUSIONS: Our data showed the biodistribution of SPIO-shRNA molecular probe in main organs can be traced by MRI in vivo. Meanwhile, it provides important information for the effectiveness of the probe by MRI at tumor in vivo.

[Pharmacokinetics and MR imaging of SPIO-shRNA dual functional molecular probe in vivo].

In this study, we investigated the pharmacokinetics parameters of SPIO-shRNA dual functional molecular probe and observed the main organ distribution by MRI in vivo. Eighteen New Zealand white rabbits were randomly divided into three groups and injected intravenously with different doses of SPIO-shRNA molecular probe, respectively. The blood samples were collected to analyze the pharmacokinetic parameters by measuring the iron content at 30 minutes before and after the injection. Twenty-four Kun Ming (KM) mice were randomly divided into 4 groups: the control group was injected intravenously with physiological saline 200 µL per mouse via the tail vein, the other 3 groups were injected intravenously with different doses of SPIO-shRNA molecular probe. MRI observation was performed in 24 hours, and the liver, spleen, kidney, brain and muscle were collected for iron quantification with Prussian blue staining to determine distribution of the SPIO-shRNA molecular probe in the main organ in vivo. Our results suggest that the molecular probe blood half-life is more than 3 hours. The data of MRI suggest the probe was distributed in liver and spleen, and the MRI signal was reduced with the increase in probe's doses (P < 0.05). The results of Prussian blue staining confirmed the results of MRI. Most of the probe could escape the phagocytosis of mononuclear phagocyte system. Our data provide the pharmacokinetic and distribution of SPIO-shRNA molecular probe in organs. Meanwhile, it suggests the choice of the time and dose of probe for MR imaging of tumor in vivo.

[Optimal concentration of superparamagnetic iron oxide-short hairpin RNA dual functional molecular probe transfected into ovarian cancer cells in vitro].

OBJECTIVE: To explore the effects of superparamagnetic iron oxide-short hairpin RNA (SPIO-ShRNA) dual functional molecular probes of different concentrations on morphology and biological behavior of ovarian cancer SKOV3 cells in vitro. METHODS: The dual functional molecular probes at an iron concentration of 5, 15, 30, 45, 75, and 100 mg/L were transfected into SKOV3 cells. The transfection rate of the probe was observed by fluorescence microscope. The distribution and content of iron particles in SKOV3 cells were determined by Prussian blue staining, atomic adsorption spectrometer and electron microscopy. Cell viability was observed by cell counting kit-8 (CCK-8).The apoptosis was detected by flow cytometry. The expression of protein within the cells was detected by Western blot. The changes of the signal intensity were measured by magnetic resonance imaging (MRI). RESULTS: The SPIO-ShRNA dual functional molecular probe was uptaken in aconcentration-dependence manner within a certain range (5-30 mg/L). When the concentration of the probe was 45 mg/L, the labeling rate of the cell was close to 100%; With the increase of the concentration of probe, the cell survival rate decreased gradually. The cell survival rate of each experimental group were 94.626%±1.050%, 93.373%±1.180%, 91.700%±3.122%, 75.100%±4.362%, 72.983%±3.233%, 71.010%±2.910%,5, 15, 30 mg/L cell survival rate was not significantly decreased, the difference was not statistically significant (P=0.226, P=0.068, P=0.475); When the concentration of the probe was greater than or equal to 45 mg/L,the survival rate decreased obviously (P<0.001); Group of 45 mg/L protein expression rate was 68.905%±3.510%, When the concentration of the probe was greater than or equal to 45 mg/L, the inhibition rate of the protein expression level of epidermal growth factor receptor was obviously higher than those of 5, 15, and 30 mg/L groups, the difference was statistically significant (P<0.001, P=0.001, P=0.003, all P<0.01); the MRI displayed that the signal intensity was decreased with increasing concentrations of the probe. The signal intensity of 45 mg/L group was 165.55±4.92, compared with the blank control group(same volume of phosphate buffer saline), normal group (unlabeled ovarian cancer SKOV3 cells), 5, 15, and 30 mg/L groups , the signal intensity of 45 mg/L group decreased significantly (all P<0.001). CONCLUSION: The dual functional molecular probe can effectively transfect and specifically inhibit the expression of SKOV3 cell lines at the iron concentration of 45 mg/L, and can also be detected by MRI. The role of diagnosis and treatment of the dual functional molecular probe has been initially confirmed.

Effects of external magnetic field on the transfection rate of SPIO-shRNADual functional molecular probe into ovarian carcinoma SKOV3 cells in vitro.

OBJECTIVE: To explore the transfection rate of SPIO-shRNA dual functional molecular probe into ovarian carcinoma SKOV3 cells in external magnetic field. METHODS: Dual functional molecular probe at an iron concentration of 45 mg/L was transfected into SKOV3 cells. The cells with coexisting probe and magnetic fields were set as the intervention group,the probe-transfected cells as negative control group, and normally cultured SKOV3 without any transfection as blank control group. The transfection rate was detected by flow cytometry. Cell viability was observed by CCK-8 assay. Epidermal growth factor receptor (EGFR) expression level in SKOV3 cells was determined by real-time quantitative PCR and Western blot analysis. The signal intensity was measured by magnetic resonance imaging (MRI). RESULTS: The transfection rate of the intervention group was (79.20 ± 3.31)%, which was significantly higher than that of negative control group (P=0.001). Compared with the negative control group,the cell viability of the intervention group significantly decreased (P=0.011), protein and mRNA expression levels of EGFR in the intervention group were significantly decreased (both P<0.05). The signal intensity on T2(*)WI in the intervention group also significantly decreased (P=0.0004). CONCLUSION: The external magnetic field can improve the transfection efficiency SPIO-shRNA dual functional molecular probe into ovarian carcinoma SKOV3 cells.