Activating transcription factor 6 contributes to cisplatin­induced ototoxicity via regulating the unfolded proteins response.

As a broad-spectrum anticancer drug, cisplatin is widely used in the treatment of tumors in various systems. Unfortunately, several serious side effects of cisplatin limit its clinical application, the most common of which are nephrotoxicity and ototoxicity. Studies have shown that cochlear hair cell degeneration is the main cause of cisplatin-induced hearing loss. However, the mechanism of cisplatin-induced hair cell death remains unclear. The present study aimed to explore the potential role of activating transcription factor 6 (ATF6), an endoplasmic reticulum (ER)-localized protein, on cisplatin-induced ototoxicity in vivo and in vitro. In this study, we observed that cisplatin exposure induced apoptosis of mouse auditory OC-1 cells, accompanied by a significant increase in the expression of ATF6 and C/EBP homologous protein (CHOP). In cell or cochlear culture models, treatment with an ATF6 agonist, an ER homeostasis regulator, significantly ameliorated cisplatin-induced cytotoxicity. Further, our in vivo experiments showed that subcutaneous injection of an ATF6 agonist almost completely prevented outer hair cell loss and significantly alleviated cisplatin-induced auditory brainstem response (ABR) threshold elevation in mice. Collectively, our results revealed the underlying mechanism by which activation of ATF6 significantly improved cisplatin-induced hair cell apoptosis, at least in part by inhibiting apoptosis signal-regulating kinase 1 expression, and demonstrated that pharmacological activation of ATF6-mediated unfolded protein response is a potential treatment for cisplatin-induced ototoxicity.

Identification of T-cell exhaustion-related gene signature for predicting prognosis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumour with a poor prognosis in adults. Identifying biomarkers that can aid in the molecular classification and risk stratification of GBM is critical. Here, we conducted a transcriptional profiling analysis of T-cell immunity in the tumour microenvironment of GBM patients and identified two novel T cell exhaustion (TEX)-related GBM subtypes (termed TEX-C1 and TEX-C2) using the consensus clustering. Our multi-omics analysis revealed distinct immunological, molecular and clinical characteristics for these two subtypes. Specifically, the TEX-C1 subtype had higher infiltration levels of immune cells and expressed higher levels of immune checkpoint molecules than the TEX-C2 subtype. Functional analysis revealed that upregulated genes in the TEX-C1 subtype were significantly enriched in immune response and signal transduction pathways, and upregulated genes in the TEX-C2 subtype were predominantly associated with cell fate and nervous system development pathways. Notably, patients with activated T-cell activity status in the TEX-C1 subgroup demonstrated a significantly worse prognosis than those with severe T cell exhaustion status in the TEX-C2 subgroup. Finally, we proposed a machine-learning-derived novel gene signature comprising 12 TEX-related genes (12TexSig) to indicate tumour subtyping. In the TCGA cohort, the 12TexSig demonstrated the ability to accurately predict the prognosis of GBM patients, and this prognostic value was further confirmed in two independent external cohorts. Taken together, our results suggest that the TEX-derived subtyping and gene signature has the potential to serve as a clinically helpful biomarker for guiding the management of GBM patients, pending further prospective validation.

Activation of Nrf2 inhibits ferroptosis and protects against oxaliplatin-induced ototoxicity.

Oxaliplatin, as a third-generation platinum-based anticancer drug, is widely used in tumor therapy of many systems. Clinically, oxaliplatin has a number of serious side effects, most notably neuropathy and ototoxicity. The degeneration of cochlear hair cells is the main reason for the hearing loss caused by platinum-based drugs. However, the mechanism of oxaliplatin-induced cochlear hair cell death remains unclear. Ferroptosis is a novel cell injury pattern triggered by the accumulation of iron hydroperoxides in lipids and dependent on the participation of iron ions, which plays an important role in a variety of diseases. Whether ferroptosis is involved in oxaliplatin-induced ototoxicity has not been reported. In this study, we observed that oxaliplatin treatment resulted in lipid peroxidation and reactive oxygen species (ROS) accumulation in OC1 cells, which may be an early alteration in the occurrence of ferroptosis. Additional treatment with ferroptosis inducer or inhibitor significantly aggravated or ameliorated oxaliplatin-induced cytotoxicity. Similarly, inhibition of ferroptosis also protected cochlear hair cells against oxaliplatin-induced injury. In addition, the expression of nuclear factor erythroid 2-related factor2 (Nrf2) and heme oxygenase-1 (HO-1) was significantly increased after oxaliplatin treatment, and treatment with the Nrf2 agonist, resveratrol, dramatically attenuated cochlear hair cell damage induced by oxaliplatin. Activation of Nrf2 significantly decreased the expression of iron regulatory protein 2 (IRP-2) and reversed the expression of glutathione peroxidase 4 (GPX4). Collectively, our results demonstrated that activation of Nrf2 alleviates oxaliplatin-induced cochlear hair cell damage by inhibiting ferroptosis, which may be a new mechanism of oxaliplatin-induced ototoxicity.

Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils.

RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX­mCCR3­1+2+3+4­shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co­transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3­(4,5­dimethylthiazol­2­yl)­5­(3­carboxymethoxyphenyl)­2­(4­sulfophenyl)­2H­tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX­mCCR3­1+2+3+4­shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

Effect of RNA interference therapy on the mice eosinophils CCR3 gene and granule protein in the murine model of allergic rhinitis.

OBJECTIVE: To observe the clinical manifestations of allergic rhinitis mice and the expression changes of the eosinophils CCR3 and the granule protein mRNA in the bone marrow, peripheral blood and nasal lavage fluid. METHODS: Twenty-four BALB/c mice were randomly divided into the control group, PBS therapy group, siRNA therapy group and the CCR3 siRNA therapy group (n=6). Allergic rhinitis model were sensitized and stimulated by ovalbunfin, and CCR3 siRNA therapy group were administered with CCR3 transnasally before stimulated. The levels of the eosinophils CCR3, MBP, ECP and EPO in bone marrow, peripheral blood and nasal lavage fluid were detected by RT-PCR. RESULTS: Compared to the control group and CCR3 siRNA therapy group, the nasal mucosa of the PBS therapy group and siRNA therapy group developed epithalaxy, goblet cells hyperplasia, squamous epithelium metaplasia, epithelium necrosis, lamina propria and submucosa gland hyperplasia, vasodilatation, tissue edema, and the characterized eosinophil infiltration. RT-PCR indicated that the CCR3 mRNA, MBP, ECP and EPO expression in bone marrow, peripheral blood and nasal lavage fluid of the CCR3 siRNA therapy group was lower than the PBS therapy group and siRNA therapy group (P<0.05). CONCLUSIONS: The RNA interference therapy to CCR3 by local administration pernasal can suppress the process of the development, migration and invasion of the allergic rhinitis eosinophil, thus can reduce the effect of eosinophils and then reduce the inflammation effect of the allergic rhinitis. It may be a new treatment for respiratory tract allergic inflammation.

[Construction and identification of mouse eosinophils CCR3 gene RNA interference lentiviral vector].

OBJECTIVE: Through construction of a lentiviral expression vector of chemokine receptor 3 (CCR3)RNA interference (RNAi) of mouse, to further study the function of CCR3 gene on eosinophils. METHODS: Focused on the CCR3 gene sequences, RNAi target sequences were designed, then the target sequences of Oligo DNA were synthesized and annealed to double stranded DNA, which was subsequently connected to pLVX-shRNA2-m vector digested by MluI, SacI, EcoRI, HindIII, BamHI and Xho I, short hairpin RNA lentiviral vectors were constructed. Short hairpin RNA lentiviral vectors were constructed. 293T cells and eosinophils were transfected by shRNA lentiviral vector, and virus titer was determined. The expression of the CCR3 gene in eosinophils was identified by quantitative-PCR. RESULTS: The lentiviral vector of shRNA-mCCR3-oligonucleotide chain was inserted correctly. Infection efficiency of 293T cells observed under fluorescence microscope was more than 90%, the virus titer was 4×10(8) TU/ml. CCR3 interference rate was 86.7%. CONCLUSION: A lentiviral vector of CCR3-gene RNAi was constructed successfully by the genetic engineering technology, and it provides a condition for further research in vitro and vivo.

Functional outcome of en bloc resection and osteoarticular allograft reconstruction with locking compression plate for giant cell tumor of the distal radius.

BACKGROUND: Giant cell tumors of the distal radius at Campanacci grade III are particularly challenging to treat. We have treated 15 cases of giant cell tumor of the distal radius by en bloc excision and osteoarticular allograft reconstruction with locking compression plate (LCP). The purpose of this study was to assess the intermediate outcomes of all patients treated with this surgery. METHODS: From July 2002 to January 2009, we followed up 15 patients with giant cell tumors of the distal radius who were treated with en bloc excision and osteoarticular allograft reconstruction with LCPs that were long enough to approach the distal end of the allograft. All of the cases were evaluated based on clinical and radiologic examinations, the passive range of motion of the wrist joint, complications, Mayo wrist score, and short form (SF)-36. RESULTS: The clinical follow-up time after reconstruction averaged 5.2 years. The mean resected length of the radius was 8.1 cm. One patient had tumor recurrence in the soft tissues after 3 years (recurrence rate 6.67 %). No patient had allograft bone fracture, nonunion, or metastases. Subchondral bone alterations and joint narrowing were present in all cases, with 1 patient suffering from the pain, but the pain could be endured without the need for analgesics. The average range of motion of the wrist was 46.7° of dorsiflexion, 33.3° of volar flexion, 61.3° of supination, and 72.3° of pronation. The mean Mayo wrist score was 70 and the mean modified SF-36 score was 71. CONCLUSIONS: En bloc excision and osteoarticular allograft reconstruction with an appropriate LCP for a Campanacci grade III giant cell tumor of the distal radius result in a reasonable functional outcome at intermediate follow-up evaluation. This method can excise the tumor integrally with a low rate of recurrence, good function, and a satisfactory range of motion.

[Effect of intranasal glucocorticoid on the pathological change of nasal mucosa in rats with allergic rhinitis.].

OBJECTIVE: To investigate the influence of inhaled glucocorticoid on the pathological change of the nasal mucosa in allergic rhinitis. METHODS: One hundred and eighty Sprague-Dawley (SD) rats were selected. According to the random number table, these animals were randomly divided into two groups: the control Group A and the experimental group. There were 60 rats in Group A and 120 rats in experimental group. First of all, the rats in experimental group were sensitized by intra-peritoneal injection with ovalbumin (OVA), enhanced and local stimulated. Next, the rats in the experimental group were randomly divided into B and C groups. The number of rats in each group was 60. Group B still had the intranasal dropping on each side with OVA in the same volume and concentration twice a week. The rats in Group C also had the intranasal dropping on each side with OVA in the same volume and concentration twice a week. But, at the same time, these animals had fluticasone propionate (FP) nasal spray each side 50 microl/per day. While doing intra-peritoneal injection with physiological saline in the same volume and intranasal dropping on the rats in normal Group A. Ten rats from each group were randomly selected to be killed at the end of first, second, fourth, eighth, twelfth and sixteenth weeks after treatment. One from the ten rats in each group was used for micro-vascular casting of nasal mucosa, and the remaining nine were used for pathological examination. RESULTS: The model of the rats in experimental group was established successfully. After allergen stimulation, the nasal mucosa showed metaplasia of the goblet cells, epithelial denudation, inflammatory cells infiltration especially eosinophils, hyperplasia of the number of gland and density of micro-blood vessels. The capillary became netted. Cilia of epithelial shed to different extent and were uneven, and the layers of reticular formation of basal membrane became thick, and collagen deposition and fabric hyperplasia were seen under the electron microscope. In Group B, due to continuously contact with allergen, the mucosa remodeling enhanced. In Group C, glucocorticoid controlled the symptoms of allergic rhinitis better, but the cilia of epithelial shed, metaplasia of the goblet cells, inflammatory cells infiltration, hyperplasia of gland and micro-blood vessels, collagen deposition and fabric hyperplasia also could be seen in rat nasal mucosa. CONCLUSIONS: The pathological change of the nasal mucosa was found in allergic rhinitis. If the allergen was continuously contacted, the pathological change aggravated. Glucocorticoid could control the symptoms of allergic rhinitis better and reverse the mucosa pathological change to some extent, but it could not retro-converse or repair the nasal mucosa while the irreversibile change had occurred.

[A study of hepatitis B virus subtypes in patients chronically infected with genotype B or C of hepatitis B virus in Guizhou].

OBJECTIVE: To investigate hepatitis B virus (HBV) subtypes in patients chronically infected with genotype B or C of hepatitis B virus in Guizhou and to study the relationship between the subtypes and the progression of their liver diseases. METHODS: Using PCR, 309 bp gene fragments in the HBV p region were amplified. The products of PCR were digested by VspI, NciI, BstEII and subjected to agarose gel electrophoresis. The subtypes of C1 and C2 were detected by restriction fragment length polymorphism (RFLP). B subtype was determined by direct sequencing of PCR product. One hundred seventy-eight patients with genotype B or C HBV infection in Guizhou, including 50 asymptomatic carriers (ASC), 100 chronic hepatitis (CHB), 14 liver cirrhosis (LC), and 14 hepatocellular carcinoma (HCC) patients were examined. The relationship between HBV C subtypes and the progression of their liver diseases was studied by analyzing the HBeAg positivity, HBV DNA loads and ALT levels of the patients. RESULTS: Of 84 patients with HBV genotype C, 27 (32.14%) and 56 (66.67%) were subtype C1 and C2, respectively. In 94 genotype B, 93 (98.94%) were subtype Ba and only one was subtype Bj. Subtype C1 showed a trend of gradual decrease from ASC and CHB to LC/HCC groups. In contrast, subtype C2 showed a gradual increase (trend) in the same order. The HBeAg positivity was significantly lower in subtype C1 than that in subtype C2. The ALT levels and HBV DNA loads were higher in patients with subtype C2 than those in subtype C1, however no statistical significance was found in these primes (t=0.95, 0.79). CONCLUSION: Subtype Ba is major and subtype C2 is more common in Guizhou. The distribution of subtype C1 and C2 are different in various stages of liver disease. The PCR-RFLP method is simple and accurate and can be used in a large-scale survey.

[Study on the distribution of hepatitis B virus precore and basic core promoter mutations in Guizhou area].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) precore A1896 and basic core promoter (BCP)T1762/A1764 mutations in Guizhou area. METHODS: 482 patients with chronic HBV infection, belonging to 4 nationalities, including 225 asymptomatic carriers (ASC), 158 chronic hepatitis (CH), 57 liver cirrhosis (LC), 42 hepatocellular carcinoma (HCC), from 4 areas of Guizhou province were examined. HBV A1896 and T1762/A1764 mutations were determined by direct sequencing and restriction fragment length polymorphism (RFLP). HBV genotypes were determined by PCR-RFLP based on S gene. The relationship among these mutations and genotype and the progression of liver disease were studied by multi-normal logistic regression analysis. RESULTS: A1896 and T1762/A1764 mutations were detected 23.03% and 29.67% among 482 patients. These mutations were more prevalent in Hans than in Dong, Miao and Buyi minorities (P < 0.01, respectively). The mutations of A1896 and T1762/ A1764 were more commonly seen in HBeAg negative than in HBeAg positive patients (P < 0.01, respectively). The mutation of T1762/A1764 was significantly higher in genotype C than in genotype B (P < 0.01). There were significantly statistical differences in the detective rate of A1896 and T1762/ A1764 mutations between patients with HCC, LC and CH, ASC. The distribution of these mutations in Guiyang (31.79% and 41.06%) was higher than in Zunye (10.94%, 14.06%), Duyun (8.64%, 11.11%) or Kaili (2.86%, 2.86%). However, there was no statistical difference by multi-normal logistic regression analysis after controlling the influence of HBeAg statu, genotype and clinical types. CONCLUSION: The distributions of A1896 and T1762/A1764 mutations were different in some nationalities of Guizhou province. The mutation of T1762/A1764 was more commonly seen in genotype C than in genotypr B. These mutations were closely related to progression of chronic liver diseases. Hepatitis B virus; Genotype; Restriction fragment length polymorphism

[Investigation on virus genotype in patients infected with hepatitis B virus in four cities of Guizhou].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) genotype in Guizhou and to study the relationship between the genotype and the progression of liver disease. METHODS: 786 patients with chronic HBV infection, from 4 cities of Guizhou, including 346 asymptomatic carriers (ASC), 313 chronic hepatitis (CH), 77 liver cirrhosis (LC), 50 hepatocellular carcinoma (HCC) were examined. HBV genotype was determined by restriction fragment length polymorphism analysis and the subtypes were determined by direct sequencing of PCR product in 94 patients with HBV B genotype, the relationship between HBV genotype and the progression of liver disease was studied by multifactor analysis such as HBeAg positivity, HBV DNA load and ALT level. RESULTS: Of the 786 patients, 7 (0.89%), 497 (63.23%), 275 (34.99%), and 7 (0.89%) belonged to genotype A, B, C, D, respectively. There was statistically significant difference in the distribution of genotype B among Kaili (96.04%), Zunyi (78.79%), Duyun (64.52%) and Guiyang (53.14%) (P< 0.01). Genotype C was more prevalent in Guiyang than in other three cities (P < 0.01, or P < 0.05). Out of 94 genotypes B, 93 (98.94%) belonged to subtype Ba, only one was subtype Bj. There were statistically significant difference in the distribution of genotype B and C among various stage of liver disease (P < 0.05 or P < 0.01). Genotype B showed a gradual decrease from ASC, CH, LC to the HCC group while in contrast, genotype C showed a gradual increase in the same order. The ALT levels and the mean age were significantly higher and older in patients with genotype C than those in genotype B (P < 0.01 or 0.05). The HBeAg positivity was significantly lower in genotype C than that in genotype B (P < 0.025). CONCLUSION: Data showed that there were genotype A, B, C and D existing in Guizhou. Genotype B was the major one but genotype C was more commonly seen. In genotype B, subtype Ba appeared to be predominant. The geographic distribution of genotype B and C were different in some cities of Guizhou. Compared to genotype B, genotype C was associated with the development of more severe liver damage.