An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.

CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.

Characterization of thyroid metastasis from clear cell renal cell carcinoma on ultrasonography: a report of three cases and literature review.

Introduction: Thyroid metastasis from clear cell renal cell carcinoma (ccRCC) is relatively rare, so ultrasound doctors lack experience with the disease, which can easily lead to misdiagnosis. We describe three cases of thyroid metastasis from ccRCC detected 12, 8, and 7 years after nephrectomy. Case presentation: The first patient, a 78-year-old woman, was admitted to our institution for hoarseness and progressive dyspnea. Ultrasonography revealed bilateral thyroid nodules and abnormal cervical lymph nodes. Fine-needle aspiration biopsy (FNAB) and core needle biopsy (CNB) of the thyroid was nondiagnostic. The other two patients, a 54-year-old man and a 65-year-old man, were admitted to our institution for a goiter pressing on the trachea. In each case, ultrasonography revealed a partially cystic nodule of the left lobe of the thyroid gland. Histological examination of three patients after thyroidectomy showed thyroid metastasis from ccRCC. Discussion/Conclusion: For patients with a history of ccRCC, long-term follow-up and routine thyroid ultrasonography should be performed. If a new thyroid nodule is found during the examination, metastases should be highly suspected. FNAB should be performed, even if benign ultrasound features seem to be in evidence. If the diagnosis of FNAB is incorrect and inconclusive, CNB should be performed.

Lenalidomide attenuates IMQ-induced inflammation in a mouse model of psoriasis.

Psoriasis is a type of chronic autoimmune-mediated inflammatory skin condition in which clinical manifestations are characterized by erythema and scaly changes, with complex pathogenesis and ease of relapse, and it is difficult to cure. Lenalidomide (Len) is a structural analog of thalidomide, which belongs to the second generation of immunomodulators and has the functions of tumor killing, immune regulation, anti-angiogenesis and regulation of the myeloma microenvironment. In the current experiment, we investigated the therapeutic effect of transdermal application of Len on the pathological changes of imiquimod (IMQ)-induced skin irritations and inflammation in psoriatic-like mice. The in vivo results revealed that Len nanoemulsion-based gels markedly reduced the IMQ-induced Psoriasis Area Severity Index (PASI) score, spleen-to-body weight index and CD4 protein expression in the derma of mice and improved IMQ-induced skin inflammatory cell infiltration. Transcriptome sequencing was intended to obtain the differentially expressed genes among the skin of Con mice and the skin of IMQ mice, and then, the GO enrichment classification and KEGG pathway analysis of the significant genes was executed to obtain major signaling pathways in the pathogenesis of the psoriasis mouse model. It was found that the PI3K/AKT signaling pathway was a major pathway in the pathogenesis of psoriasis in a mouse model induced by IMQ. The immunohistochemical results confirmed that Len could modulate the protein expression of AKT and NF-κB in skin. In conclusion, the protective effect of transdermal administration of Len may be related to the inhibition of the PI3K/AKT signaling pathway and its downstream NF-κB pathway against IMQ-induced psoriasis in mice.

A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction.

MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans-cleavage activity of Cas12a for further signal amplification. More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.

Regulation effects of hydrogen sulfide on ascorbate-glutathione cycle in naked oat leaves under saline-alkali stress.

Saline-alkali stress is one of the common abiotic stresses for plants. Hydrogen sulfide (H2S), as a gas signal, plays an important role in driving the responses of plants to saline-alkali stress. To explore the regulating effects of H2S on the ascorbate (AsA)-glutathione (GSH) cycle in naked oat (Avena nude) under saline-alkali stress, we used sodium hydrogen sulfide (NaHS) as donor of exogenous H2S and hydroxylamine (HA) as H2S synthesis inhibitor to examine the effects of H2S on plant growth, leaf reactive oxygen species, membrane lipid peroxidation, and antioxidants and key enzymes in the AsA-GSH cycle in "Dingyou 9" variety of naked oat under saline-alkali mixed stress. Results showed that spraying 50 µmol·L-1 NaHS could alleviate the inhibition of 50 mmol·L-1 saline-alkali mixed stress on the growth of naked oats, reduce the content of superoxide anions, H2O2, malondialdehyde, oxidized ascorbate (DHA), glutathione (GSH), and oxidized glutathione (GSSG) in leaves of naked oat under saline-alkali mixed stress, increase the ratio of AsA/DHA and GSH/GSSG, but did not affect the content of reduced ascorbic acid (AsA). Spraying NaHS significantly increased the activities of key enzymes, L-galactose dehydrogenase (GalDH) and L-galactono-1, 4-lactone dehydrogenase (GalLDH), for AsA synthesis pathways in naked oat leaves under salt-alkali mixed stress, as well as monodehydroascorbate reductase (MDHAR) in the AsA-GSH cycle, and decreased the activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), but did not affect the activities of ascorbate oxidase (AO) and glutathione reductase (GR). The addition of HA partially or completely relieved those aforementioned effects. Our results indicated that H2S could increase the efficiency of AsA-GSH cycle by promoting the synthesis of AsA and enhancing the activity of MDHAR, and reduce the oxidative damage of saline-alkali stress to naked oats.

LncRNA MEG3 suppresses PI3K/AKT/mTOR signalling pathway to enhance autophagy and inhibit inflammation in TNF-α-treated keratinocytes and psoriatic mice.

BACKGROUND: Psoriasis is a common chronic inflammatory skin disorder that causes patches of thick red skin and silvery scales and affects 1-3% of the population, which reduces patient's quality of life. Understanding the pathogenesis of psoriasis is crucial for developing novel therapeutic strategies. METHODS: HaCaT and NHEK cells were treated with TNF-α in vitro. A mouse model of psoriasis was established by topical imiquimod application on back skin. LncRNA MEG3 was cloned into the pcDNA3.1 vector and transfected in TNF-α-treated HaCaT and NHEK cells to overexpress its expression. Liposome-encapsulated pcDNA3.1-MEG3 was injected into imiquimod-treated mice via tail vein. RT-qPCR and western blot assays were used to examine the expression of lncRNA MEG3, IL-6, IL-8, IFN-γ, IL-1ß, LC3, Beclin 1, p62, p-p65, p65, NLRP3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR respectively. The secretion of IL-6, IL-8, IFN-γ and IL-1ß was determined using ELISA assay. Immunofluorescence and immunohistochemistry methods were performed for analyzing the expression of LC3 and NLRP3 in cells and skin tissues respectively. LY294002 was used to block the PI3K/AKT/mTOR signalling. MTT assay was applied to test the toxicity of LY294002 to HaCaT and NHEK cells. RESULTS: LncRNA MEG3 expression levels were downregulated in TNF-α-treated HaCaT and NHEK cells and skin tissues of psoriatic mice model. TNF-α treatment enhanced inflammation and suppressed autophagy in HaCaT and NHEK cells, which were largely reversed by overexpression of lncRNA MEG3. Autophagy puncta and NLRP3 inflammasome assembly showed the same patterns with the expression of inflammation and autophagy markers in TNF-α-treated HaCaT and NHEK cells with or without lncRNA MEG3 overexpression. TNF-α-induced activation of the PI3K/AKT/mTOR signalling was abolished by lncRNA MEG3 overexpression in HaCaT and NHEK cells. Blocking the PI3K/AKT/mTOR signalling inhibited TNF-α-induced inflammation and restored autophagy level in TNF-α-treated HaCaT and NHEK cells. Overexpression of lncRNA MEG3 suppressed inflammation, promoted autophagy and inhibited the activation of the PI3K/AKT/mTOR signalling in a mouse model of psoriasis. CONCLUSION: LncRNA MEG3 facilitates autophagy and suppresses inflammation in TNF-α-treated keratinocytes and psoriatic mice, which is dependent on the PI3K/AKT/mTOR signalling pathway. Our study enhances the understanding of psoriasis and provides potential therapeutic targets for psoriasis.

The regional distribution of melanosomes in the epidermis affords a localized intensive photoprotection for basal keratinocyte stem cells.

Human skin is a highly efficient self-renewing barrier that is critical to withstanding environmental insults. Undifferentiated keratinocyte stem cells reside in the basal layer of the epidermis and in hair follicles that continuously give rise to progenies ensuring epidermal turnover and renewal. Ultraviolet (UV) radiation is a proven cause of skin keratinocyte cancers, which preferentially occur at sun-exposed areas of the skin. Fortunately, melanocytes produce melanin that is packaged in specific organelles (termed melanosomes) that are then delivered to nearby keratinocytes, endowing the recipient cells with photoprotection. It has long been thought that melanosome transfer takes place stochastically from melanocytes to keratinocytes via an as-yet-unrecognized manner. However, recent studies have indicated that melanosomes are distributed regionally in the basal layer of the skin, affording localized intensive photoprotection for progenitor keratinocytes and stem cells that reside in the microenvironment of the basal epidermis. In this review, we summarize current knowledge about molecular and cellular mechanisms that are responsible for the selective transfer and exclusive degradation of melanosomes in the epidermis, emphasizing implications for skin carcinogenesis.

LncRNA MEG3 influences the proliferation and apoptosis of psoriasis epidermal cells by targeting miR-21/caspase-8.

BACKGROUND: It was reported that microRNA-21(miR-21) was differentially expressed in the keratinocytes of psoriasis patients, and it may influence the apoptosis and proliferation of cells. The role of lncRNA maternally expressed gene3 (MEG3), a competing endogenous RNAs of miR-21, in the progression of psoriasis remains unclear. We aimed to unfold the influence of MEG3 and miR-21 on the proliferation and apoptosis of psoriasis epidermal cells. METHODS: 50µg/L TNF-α was used to treat HaCaTs and NHEKs cells for 24 h, and then different experiments were conducted. qRT-PCR were applied for measuring the mRNA level of MEG3, miR-2, and caspase-8, and the protein expression of caspase-8 was measured with western blotting. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using MTT and colony formation assays. Dual luciferase reporter assay was applied for confirming the binding site between MEG3 and miR-21, miR-21 and Caspase-8. RESULTS: A cell model for in vitro studying the role of MEG3 in psoriasis pathophysiology was established using HaCaT and HHEKs. MEG3 was significantly down-regulated in HaCaT, HHEKs, and psoriatic skin samples. MEG3 inhibits proliferation and promotes apoptosis of Activated-HaCaT (Act-HaCaT) and Activated-HHEKs (Act- HHEK) by regulating miR-21, and the binding site between MEG3 and miR-21 was identified. We also found that miR-21 could inhibit the level of caspase-8 and identified the binding site between caspase-8 and miR-21. Some down-stream proteins of caspase-8, Cleaved caspase-8, cytc, and apaf-1 were regulated by miR-21 and MEG3. CONCLUSION: MEG3/miR-21 axis may regulate the expression of caspase-8, and further influence the proliferation and apoptosis of psoriasis keratinocyte, Act-HaCaT and Act- HHEK. Therefore, our findings may provide a new thought for the study of pathogenesis and treatment of psoriasis.

The role of macrophages in the differentiation process of ureteral polyps.

Objective To evaluate the role of macrophage infiltration in the differentiation process of ureteral polyps and cancers. Methods This retrospective immunohistochemical study analysed archival samples of pathologically-confirmed specimens of low- and high-grade ureteral cancer, ureteral papilloma and ureteral polyps. The samples were immunohistochemically stained for cluster of differentiation (CD)4, CD8, CD16, CD25, CD56 and CD68 using immunofluorescence in order to identify different T-lymphocyte populations and macrophages. Results A total of 70 specimens were included in the analysis: 21 specimens of ureteral cancer, 17 specimens of ureteral papilloma, and 32 specimens of ureteral polyps. The largest proportion of CD4+CD25+ regulatory T cells was observed in the low-grade ureteral cancer group and almost none were observed in ureteral papillomas. The largest proportion of CD8+ cytotoxic T-lymphocytes was observed in the ureteral polyps. The largest proportion of CD56+ natural killer cells was detected in the ureteral polyps, with very low levels observed in the other three groups. The largest proportion of CD16+CD68+ macrophages was observed in the high-grade ureteral cancer group, which was significantly higher than that observed in the ureteral papillomas. Conclusions This study revealed that CD16+CD68+ macrophages appear to participate in ureteral neoplastic transformation.

HDAC5 Inhibits Hepatic Lipogenic Genes Expression by Attenuating the Transcriptional Activity of Liver X Receptor.

BACKGROUND/AIMS: Liver X receptor (LXR), a member of the nuclear receptor superfamily, is known to induce the expression of SREBP-1c and ChREBP, two master regulators of hepatic lipogenesis. Histone deacyetylases (HDACs) have been shown to play critical roles in glucose and lipids metabolism. However, the exact role of HDAC5 in lipogenesis remains elusive. METHODS: mRNA and protein levels of HDAC5 were analyzed by quantitative real-time PCR and Western blots in high-fat-diet-induced and leptin receptor deficiency-induced obese mice. HDAC5 was overexpressed or depleted in HepG2 cells, followed by analysis of cellular triglycerides contents. Quantitative real-time PCR was used to detect the expression levels of lipogenic genes. Luciferase reporter assay was used to determine the regulation of HDAC on the transcriptional activity of LXR. Co-immunoprecipitation experiment was used to determine the interaction between HDAC5 and LXR. RESULTS: We found that mRNA and protein expression levels of hepatic HDAC5 were reduced in high-fat-diet-induced and leptin receptor deficiency-induced obese mice. In vitro studies further demonstrated that knockdown of HDAC5 promoted cellular triglycerides accumulation, accompanied with up-regulation of lipogenic genes. At the molecular level, HDAC5 was shown to interact with LXR, thereby attenuating its transcriptional activity. CONCLUSION: Overall, our data suggest that hepatic HDAC5 is an important regulator of lipogenesis.

Asymmetric stem-cell division ensures sustained keratinocyte hyperproliferation in psoriatic skin lesions.

Excessive expansion of the transit-amplifying (TA) cell compartment is a distinct morphological characteristic of psoriatic epidermal hyperplasia. In order to examine the activation of basal stem cells and how they replenish such an enlarged compartment of TA cells in psoriatic epidermis, we utilized a BrdU labeling method to monitor mitotic stem cells in a mouse model of psoriasiform dermatitis, which was induced by imiquimod. Our results showed that perpendicular and parallel cell division characteristics of dividing stem cells existed in the inflamed epidermis. When we analyzed template­DNA strand segregation in trypsin-dissociated human psoriatic keratinocytes using BrdU pulse-chase labeling, we found that the percentage of asymmetric segregation of BrdU was significantly increased in the cell pairs of psoriatic epidermal cells compared with normal epidermal cells. Furthermore, we also examined the effects of both interleukin (IL)-17A and IL-22 cytokines on the differentiation status of cultured human keratinocytes. The results indicated that both cytokines had synergistic effects on passage-one epidermal cell sheets derived from skin explants and also on cultured keratinocytes, were involved in the maintenance of the undifferentiated stem cell phenotype, and these results suggest an efficient mechanism for preventing the premature loss of basal stem-cell pools in the pro-inflammatory cytokine-enriched milieu of the psoriatic epidermis. Our findings suggest that inhibition of hyperactive stem cells represents a potential therapeutic target to combat recalcitrant epidermal hyperplasia in psoriasis.

The technique comparison of brachial plexus blocks by ultrasound guided with blocks by nerve stimulator guided.

OBJECTIVE: Brachial plexus perineural blocks provide specific analgesia for upper limb surgery. We present our experience with ultrasound-guided supraclavicular brachial plexus perineural blocks for distal upper limb surgery. Although single-injection ultrasound-guided supraclavicular blocks have been reported, little is known about the advantages using this approach compared with nerve stimulator guided. METHODS: There were 60 patients who underwent upper limb surgery for orthopedic trauma and received a supraclavicular brachial plexus anesthesia. 30 patients (U-group) were injected by an ultrasound-guided technique with the needle tip remaining under direct vision. 30 patients (NS-group) were inserted by nerve stimulator guided. Recorded the onset time, puncture times, pains cases with tourniquet in each group. Compared the difference between two groups. RESULTS: In U-group, all cases had successful perineural injection. Most of them, effect of anesthesia was fast onset and needed insert only once. No pains were reported under using tourniquet. There were no vessel punctures or other direct procedure-related complications. In NS-group, most injections were successful, but slow onset and needed multiply insert needle. 5 patients said pains under using tourniquet when surgery started and had to add opioid by vein. One patients' lung were puncture and result in pneumothorax. One patient's was intravascular injection. CONCLUSIONS: Supraclavicular brachial plexus perineural insertion using ultrasound guidance is feasible and almost have no complications, deserves further study with a randomized controlled trial comparing this relatively new technique with only using nerve stimulator.

Efficacy and safety between temozolomide alone and temozolomide-based double therapy for malignant melanoma: a meta-analysis.

Temozolomide (TMZ) has received much attention, notably in the treatment of malignant glioma and malignant melanoma. The objective of this study was to compare the clinical efficacy and safety of TMZ alone and TMZ-based combination drug therapy in patients with melanoma. Using "temozolomide" as a keyword combined with "melanoma" and "randomized controlled trials" as Medical Subject Headings, the following electronic databases were searched: the Cochrane library, MEDLINE, EBSCO, EMBASE, Ovid, cNKI, and cBMDisc. The evaluating indicators were overall response rate (ORR), 1-year survival rate, and several of the most frequent adverse events. Five randomized controlled trials met our criteria and were included in the meta-analysis, with a total of 703 participants (309 patients received TMZ alone, and 394 patients received combined regimens). The meta-analysis showed that the ORR for TMZ-based drug therapy was higher than TMZ alone [relative risk (RR) = 1.44; 95% confidence interval (CI), 1.06-1.95], but the 1-year survival rate was not significantly different between the two groups (RR = 1.13; 95% CI, 0.92-1.40). These results suggested that the impact of these increased response rates was not translated into a survival benefit. Moreover, we found no difference in the incidence of adverse events analyzed. The currently available evidence showed that the TMZ-combination therapy may moderately improve the response rate, but there was no corresponding increased toxicity. Future large-scale, high-quality, placebo-controlled, double-blind trials are needed.

The multi-lineage differentiation and angiogenesis ability of adipose-derived adult mesenchymal stem cells.

BACKGROUND: To investigate the in vitro multi-lineage differentiation of adipose-derived adult stem cells and their ability to differentiate into endothelial cells. METHODS: Adipose-derived adult stem cells were isolated for detection of the immune phenotype, cell doubling time, cycle, and induction of endothelial cell differentiation in vitro. The expression of endothelial cell-specific surface markers was measured immunocytochemically. RESULTS: Adipose-derived adult stem cells have multi-lineage differentiation potential and can differentiate into endothelial cells in vitro. CONCLUSIONS: Adipose-derived adult stem cells have the same differentiation ability with those derived from the bone marrow, as both can differentiate into endothelial cells. These findings have opened up the prospect of adipose-derived adult stem cells in angiogenesis therapy.

[Witchcraft medicine and folklore in Wushierbingfang ('Prescriptions for fifty-two diseases')].

One important characteristic of early stage of TCM is the intermixture of witches medicine and folklore. A few witch prescriptions in Wushierbingfang ('Prescriptions for fifty-two diseases') indicated the residual traces of the mixture of witch and medicine in the medical literatures. The witch prescriptions recorded in Wushierbingfang ('Prescriptions for fifty-two diseases') could be divided into supplication, Yu-step, exorcism, Nuo ritual and peach wood charms etc. Witchcraft developed into folklore and the application of witchcraft sometimes manifested as the form of folklore, which were also reflected in the records of ('Prescriptions for fifty-two diseases').

[The effect of cold stress on DNA oxidative damage of lung in chicken].

AIM: To explore the influence of cold strss on DNA oxidative damage of lung in chicken. METHODS: Took 15-day-old healthy chicks as the experimental object, carried on the cold stress (12 +/- 1 degrees C) to process. Detected the change of the MDA content, SOD and GSH-Px activity of the lung, and performed KCl-SDS precipitation method and fluorescence detection method to identify the influence of cold strss on DNA-protein crosslinks (DPC) and DNA-DNA crosslinks (DDC) of lung cell in different time. RESULTS: The results were as follow: with the time lapsing during acute cold stress, MDA content gradually increased, the SOD and GSH-Px activity of the lung increased compared with their control group at each stress time point, and the lung cell DPC and DDC coefficient were all gradually increased with the time lapsing. CONCLUSION: Cold stress could bring about destruction in the lung tissue oxidation-antioxidant balance, and causes the oxidation damage of DNA.

[Effects of glutamine on the colon of mice subjected to colitis gravis].

AIM: To investigate the effects of glutamine on the colonic mucosa of mice subjected to colitis gravis. METHODS: 64 Kunming mice were divided randomly into 4 groups (n=16): healthy group: animals not subjected to colitis; model group: animals subjected to colitis gravis but without glutamine supplementation; low-Gln group: animals subjected to colitis gravis and with low dose of glutamine supplementation; high-Gln group: animals subjected to colitis gravis and with high dose of glutamine supplementation. Animals belonging to the control, the low-Gln, the high-Gin groups were subjected to coloclysis by HAC to be colitis gravis animals. When the models were established, the healthy and the control groups were given some isotonic Na chloride by intragastric administration. The low-Gln group and the high-Gln group were given the same volume but different concentration of glutamine(low-Gln group--2 mmol x Kg(-1) bw, high-Gln group--2 mmol x Kg(-1) bw) for 7 days. Then the mice were sacrificed, the pathohistological changes of the colon were observed, besides, the content of endotoxin in the blood serum, the level of counteracting oxidation and the activities of MPO of the colon tissue were determined. RESULTS: The glutamine lessened the pathological injures in the colon and relieved the step up of the content of endotoxin in the blood serum , the step down level of counteracting oxidation and the step up activity of MPO in the colon tissue, which were caused by colitis gravis. CONCLUSION: The glutamine can protect the colon of mice subjected to colitis gravis.

[Comparative study of mannitol-induced acute kidney impairments in patients of different ages suffering from subarachnoid hemorrhage].

OBJECTIVE: To analyze and compare the clinical characteristics of mannitol-induced acute renal function impairment in treatment of patients of different ages suffering from subarachnoid hemorrhage (SAH) for the first time. METHODS: This study was a review of 1 361 patients with available hospital records admitted to the division of neurology due to SAH from February 1989 to December 2005. Among them, 94 patients had acute renal function impairment as a result of mannitol administration. Of these patients, 35 patients were middle-aged (<60 years old) and 59 patients of old age (>60 years old). The study included the time of appearance of renal function impairment after mannitol medication, its prognosis, the administration of nephrotoxic drugs, and the dose and duration of mannitol therapy. RESULTS: In old age group, abnormal renal function and urine routine appeared in 5 days (median), and 4 days (median) respectively, and acute renal failure (ARF) was diagnosed in 5 days, and the incidence rate of ARF was 20.3% after treatment with mannitol. The respective event appeared 7 days, 11 days, 9 days and 2.8% (P<0.05 or P<0.01) respectively, in middle-aged group. The data indicated the elderly patients had poorer tolerance to mannitol, with earlier occurrence of mannitol nephrosis, poorer outcome of kidney impairment and worse prognosis. Forty-three patients (72.9%) were treated with katlex (with a median of total dosage of 400 mg) in old-aged group, 35 patients (100.0%) were treated with katlex (with a median of total dosage of 800 mg) in middle-aged group, and there was a significant difference between two groups (P<0.01). The data indicated the incidence of mannitol nephrosis was lower in patients treated with large dosage of mannitol and katlex. The mortality was 3.4% (2/59 cases) and 0% (0/35 cases) in old-aged and middle-aged groups respectively (P=0.528). CONCLUSION: The elderly patients have higher rate of mannitol-induced impairment of renal function after SAH than middle-aged patients. The data emphasize that proper combination therapy of mannitol with katlex is an effective measure in preventing renal failure.

[Regulation effect of d-alpha-tocopherol on the expression of hexokinase induced by high glucose in cultured human peritoneal mesothelium cells].

OBJECTIVE: To investigate the effect of d-alpha-tocopherol on the expression of hexokinase (HK) induced by high glucose in cultured human peritoneal mesothelium cell (HPMC). METHODS: Specimens of human omentum were obtained from consenting patient undergoing elective abdominal surgery. HPMC were isolated and subcultured by enzymatic disaggregation. Morphology and immunocytochemical method were used for identification. HPMC were divided into normal glucose group (0.1% glucose, equal to 5.5 mmol/L), high glucose group (0.5%, 1.0%, 1.5%, 2.5%, 4.25% glucose) and d-alpha-tocopherol group. After 24 h, standard G6PDH-coupled assay, temperature sensitive essay were used to detect the activity of total HK and its isozyme. Immunocytochemical staining was used for observation the intracellular location of HKII. Western blotting was used to analyze the protein expression of HKII and RT-PCR was used to detect the mRNA expression of HKII. The net glucose utilization was assayed by glucose disappearance from medium by hexokinase method. RESULTS: Primary cultured HPMC reacted positively for cytokeratin and vimentin and had numerous surface microvilli under electron microscope. High glucose induced HK activity in a dose-dependent manner and increased HKII isoform selectively. At concentration of 1.5%, 2.5% and 4.25% glucose for 24 h, the relative activity of total HK were 115.4%, 129.1% and 155.2%, respectively comparing with normal control (P < 0.05), and selectively increased HKII isoform expression. D-alpha-tocopherol blocked the activity of total HK and HKII induced by glucose. The relative activity of total HK inhibited by d-alpha-tocopherol was 82.1% (P = 0.001). After incubated with d-alpha-tocopherol, the net glucose utilization were decreased from (25.3 +/- 3.9) mmol/L to (17.3 +/- 2.1) mmol/L (P = 0.018). HKII stained light brown in cytoplasm of HPMC in normal group, accompanying with the increased concentration of glucose, the staining of HKII became strong and accumulated to nuclear. D-alpha-tocopherol made it thinning. The protein and mRNA expression of HKII were accorded with its activity. CONCLUSION: D-alpha-tocopherol inhibited the expression of HKII in activity, protein and mRNA induced by high glucose and decreased the net glucose utilization, which might become a method to improve ultrafiltration in peritoneal dialysis.