Effects of Maren Pills on the Intestinal Microflora and Short-Chain Fatty Acid Profile in Drug-Induced Slow Transit Constipation Model Rats.

Background: Slow transit constipation (STC) is becoming a common and frequently occurring disease in today's society, and it is necessary to explore the safe and effective treatment of STC. Method: Our study aimed to investigate whether the laxative effect of Maren pills (MRW) is associated with the regulation of intestinal microflora and intestinal metabolism in the colon. Loperamide hydrochloride-induced STC rats received MRW intragastrically for two consecutive weeks to evaluate the laxative effect of MRW involving the regulation of intestinal microflora, intestinal metabolism, and 5-HT signaling pathway. Intestinal microflora was detected by 16s rDNA sequencing, intestinal metabolism of short-chain fatty acids (SCFAs) was detected by HPLC, and the 5-HT signaling pathway was detected by WB, ELISA, immunofluorescence, and immunohistochemical analysis. Results: Our results revealed that the treatments with MRW increased not only the body weight, 24-h fecal number, 24-h wet fecal weight, 24-h dry fecal weight, fecal water content, and the intestinal propulsion rate but also the colonic goblet cell number, colonic Muc-2 protein expression, and colonic mucus layer thickness in the STC model rats. Moreover, MRW activated the 5-HT pathway by increasing the levels of 5-HT, 5-HIAA, 5-HT4R, CFTR, cAMP, and PKA in the colon tissue of STC rats. The 16S rDNA sequencing results showed that MRW improved the colonic microflora structure in colonic contents of STC rats, mainly by increasing Lactobacillus and decreasing Prevotella. Finally, we found that MRW regulated the SCFA metabolism in the colonic contents of the STC rats, mainly by increasing the contents of acetic acid, propionic acid, and butyric acid; the relative abundance of Lactobacillus was positively correlated with either contents of acetic acid, propionic acid, and butyric acid, and the relative abundance of Clostridium was negatively correlated. Conclusion: Our study further showed that MRW could improve constipation in STC rats, and the mechanism may be by regulating the intestinal microflora structure and improving the metabolism of SCFAs.

Diode laser combined with intradiscal vancomycin for iatrogenic discitis: an experimental study in rabbits.

OBJECTIVE: The purpose of this study was to assess the efficacy of diode laser combined with intradiscal vancomycin for iatrogenic discitis (ID) infected with Staphylococcus aureus. BACKGROUND DATA: In vivo and in vitro studies have reported that diode laser has a potential bactericidal effect on S. aureus. METHODS: The rabbit ID model was induced by injecting S. aureus into the intervertebral discs (IVDs). The animals were classified into the following groups: Group M, model; Group N, non-treatment; Group O, operation; Group V, vancomycin; Group L, laser; and Group C, laser with vancomycin. The rabbits were killed when paraplegia occurred and target IVD regions were removed for histopathological examination. RESULTS: In Group M, MRI findings revealed narrowing of the disc space, with changed T1 and T2 signals. In Group N, pathological examination showed a narrowed disc space, inflammatory changes in disc tissue, extensive erosion and hyperostosis of bony end plates, and an epidural abscess. Narrowed disc space and bone fusion were observed in Group O. In Group V, narrowed disc spaces, focal inflammatory changes of the disc tissue, and focal erosion and hyperostosis of bony end plates were seen. In Groups L and C, cavitation, inflammatory lesions, focal erosion, and hyperostosis of bony endplates were observed. However, in Group C, fibrosis was found in the nucleus region, with a smaller area of cavitation and better preservation of IVD structure than in Group L. The ID score was lowest in Group O, at 9.7 ± 0.95. The ID scores in Groups V and L were 12.2 ± 1.32 and 12.6 ± 0.97, respectively, significantly less than in Group N. The Group C ID score of 10.9 ± 0.99 was significantly less than that of Groups N, V, and L. CONCLUSIONS: High power diode laser combined with intradiscal vancomycin contained the pathological process of ID in this rabbit model.

Targeted proteomic analysis of 14-3-3sigma in nasopharyngeal carcinoma.

14-3-3sigma is a potential tumor suppressor, and loss of 14-3-3sigma expression plays an important role in carcinogenesis and metastasis. To explore the possible mechanism of 14-3-3sigma in nasopharyngeal carcinoma (NPC) invasion and metastasis, targeted proteomic analysis was performed on 14-3-3sigma-associated proteins from NPC cells. As the results, 112 proteins associated with 14-3-3sigma were identified, and four 14-3-3sigma-interacted proteins: keratin 8, epidermal growth factor receptor (EGFR), small GTP-binding protein RAB7, and p53 were confirmed by coimmunoprecipitation and Western blot analysis. The 14-3-3sigma-associated proteins could be grouped into eight clusters based on their molecule functions. Protein-protein interaction (PPI) analysis indicated that 14-3-3sigma/EGFR/keratin 8 interactions may be involved in the invasion and metastasis of NPC. 14-3-3sigma/EGFR/keratin 8 could form complexes in NPC cells. 14-3-3sigma downregulation in NPC may lead to the overexpression of EGFR and keratin 8, which increases the invasion ability of NPC cells possibly by activating the downstream signal molecules and reorganizing cytoskeleton. The data suggest that the biological functions of 14-3-3sigma in NPC are diversified, and 14-3-3sigma could inhibit the in vitro invasive ability of NPC cells possibly through 14-3-3sigma/EGFR/keratin 8 interaction.

Proteomic analysis to identify cytokeratin 18 as a novel biomarker of nasopharyngeal carcinoma.

PURPOSE: In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. METHODS: Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cytokeratin 18 in the above two tissues as well as 4 NPC cell lines was determined by Western blotting. Immunohistochemistry was also performed to detect the expression of cytokeratin 18 in 62 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. siRNA and in vitro cell invasion assay were used to check the correlation between the expression of cytokeratin 18 and invasive ability of NPC. RESULTS: Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cytokeratin 18 in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Significant cytokeratin 18 down-regulation was observed in NPC versus NNET (P = 0.000), whereas significant cytokeratin 18 up-regulation was observed in lymph node metastasis versus primary NPC (P = 0.001). In addition, cytokeratin 18 down-regulation was significantly correlated with poor histological differentiation (P = 0.000), whereas cytokeratin 18 up-regulation was significantly correlated with advanced clinical stage (P = 0.019), recurrence (P = 0.000), and regional lymph node metastasis (P = 0.001), and distant metastasis (P = 0.000). And down-regulated cytokeratin 18 expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Furthermore, survival curves showed that patients with cytokeratin 18 up-regulation had a poor prognosis (P = 0.000). Univariate analysis (Cox's proportional hazards model) showed that WHO histologic type (P = 0.025), lymph node metastasis (P = 0.007), distant metastasis (P = 0.005), recurrence (P = 0.000), and cytokeratin 18 (P = 0.000) were significantly associated with the prognosis of NPC. Multivariate analysis confirmed that lymph node metastasis (P = 0.012), distant metastasis (P = 0.009), recurrence (P = 0.006), and cytokeratin 18 (P = 0.001) were independent prognostic indicators. CONCLUSIONS: The data suggest that cytokeratin 18 is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Inactivation of 14-3-3 sigma by promoter methylation correlates with metastasis in nasopharyngeal carcinoma.

14-3-3 sigma, the downstream target of p53, is a negative regulator of cell cycle G2-M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14-3-3 sigma was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non-cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14-3-3 sigma inactivation. Methylation-specific PCR showed 14-3-3 sigma promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5-aza-2'-dC resulted in the demethylation and upregulation of 14-3-3 sigma. In tissues, 14-3-3 sigma promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14-3-3 sigma promoter was detected in NPC but not in any of adjacent NNET. RT-PCR, Western blotting, and immunohistochemistry showed that 14-3-3 sigma expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14-3-3 sigma gene. In addition, the patients with methylated 14-3-3 sigma presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14-3-3 sigma in NPC cell line 5-8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14-3-3 sigma is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis.

[Screening for differentially expressed proteins in nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis].

OBJECTIVE: To search for the differentially expressed proteins of nasopharyngeal carcinoma (NPC),and provide scientific evidence for identifying molecular biomarkers for NPC. METHODS: Laser capture microdissection (LCM) was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues (NNET). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of microdissected NPC and NNET, PDQuest software was applied to analyze 2-DE images,and the differential proteins between the 2 types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Western blot and immunohistochemistry of tissue microarray were used to detect the expression of the differential protein SCCA1 in NPC and NNET. RESULTS: 2-DE patterns of microdissected NPC and NNEC were established,and 36 differential proteins in the NPC and NNEC were identified,20 of which only expressed or up-regulated in NPC and 16 only expressed or up-regulated in NNET. The differentially expressed level of SCCA1 in the NPC and NNET was confirmed by Western blot and immunohistochemistry of tissue microarray. CONCLUSION: Thirty-six differentially expressed proteins identified in this study may be associated with the carcinogenesis of NPC,and may be candidate molecular biomarkers for NPC.

Identificating cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis.

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Identification of novel nasopharyngeal carcinoma biomarkers by laser capture microdissection and proteomic analysis.

PURPOSE: To identify novel nasopharyngeal carcinoma (NPC) biomarkers by laser capture microdissection and a proteomic approach. EXPERIMENTAL DESIGN: Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of three differential proteins (stathmin, 14-3-3sigma, and annexin I) in the above two tissues as well as four NPC cell lines was determined by Western blotting. Immunohistochemistry was also done to detect the expression of three differential proteins in 98 cases of primary NPC, 30 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of their expression levels with clinicopathologic features and clinical outcomes were evaluated. RESULTS: Thirty-six differential proteins between the NPC and NNET were identified. The expression levels of stathmin, 14-3-3sigma, and annexin I in the two types of tissues were confirmed and related to differentiation degree and/or metastatic potential of the NPC cell lines. Significant stathmin up-regulation and down-regulation of 14-3-3sigma and annexin I were observed in NPC versus NNET, and significant down-regulation of 14-3-3sigma and annexin I was also observed in lymph node metastasis versus primary NPC. In addition, stathmin up-regulation and down-regulation of 14-3-3sigma and annexin I were significantly correlated with poor histologic differentiation, advanced clinical stage, and recurrence, whereas down-regulation of 14-3-3sigma and annexin I was also significantly correlated with lymph node and distant metastasis. Furthermore, survival curves showed that patients with stathmin up-regulation and down-regulation of 14-3-3sigma and annexin I had a poor prognosis. Multivariate analysis revealed that the expression status of stathmin, 14-3-3sigma, and annexin I was an independent prognostic indicator. CONCLUSION: The data suggest that stathmin, 14-3-3sigma, and annexin I are potential biomarkers for the differentiation and prognosis of NPC, and their dysregulation might play an important role in the pathogenesis of NPC.

IL-1beta sensitizes rat intervertebral disc cells to Fas ligand mediated apoptosis in vitro.

AIM: To determine the apoptotic effect of recombinant rat Fas Ligand on rat intervertebral disc cells pre-treated with IL-1beta in vitro, and the expression of Fas in cultured rat intervertebral disc cells. METHODS: Cells were isolated from the inner annulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and divided in 5 treatment groups. IL-1beta (10 ng/mL), FasL (5, 20 ng/mL) with/without IL-1beta (10 ng/mL) pre-treatment was respectively added in Dulbeccoos modified Eagleos medium and Hamos F-12 medium with 1% fetal bovine serum. After 32 h, the cells were stained with annexin V-FITC and propidium iodide to evaluate apoptosis using flow cytometry and to analysis transcription of Fas using RT-PCR. RESULTS: Compared with control group, FasL (20 ng/mL), IL-1beta (10 ng/mL)+FasL (5 ng/mL), and IL-1beta (10 ng/mL)+FasL (20 ng/mL) induced significant apoptosis of the disc cells (P<0.01). Apoptosis was also induced by FasL 5 ng/mL (P<0.05); whereas, apoptosis was not induced by IL-1beta (10 ng/mL) (P>0.05). IL-1beta (10 ng/mL) enhanced the apoptosis-inducing effects of FasL (5 ng/mL) and FasL (20 ng/mL) in disc cells. Fas gene transcription in all groups and Fas expression in the 5 treatment groups were approximately 1.2-2.1-fold greater than control group (respectively, P<0.05). Additionally, Fas expression in FasL with IL-1beta pre-treatment groups were significantly up-regulated than in FasL groups (P<0.01). CONCLUSION: The results of this study showed disc cells pre-treated with IL-1beta increased apoptotic rate in response to FasL in vitro and provided insights to understand Fas/FasL system-mediated apoptosis in disc cells which would be enhanced due to inflammation factor in degenerative disc.

Aspirin attenuates gentamicin ototoxicity: from the laboratory to the clinic.

This article reviews recent advances in the protection from the adverse auditory or vestibular side effects associated with antibacterial treatment with aminoglycoside antibiotics. Compelling evidence from animal models suggests that reactive oxygen species are part of the initial mechanisms that trigger apoptotic and necrotic cell death in the inner ear. Consequently, antioxidants protect against aminoglycoside-induced hearing loss in animals and, importantly, they do so without compromising drug serum levels or antibacterial efficacy. While clinical studies have long confirmed the ototoxicity of aminoglycosides in human, a trial on protection was only recently reported (Sha, S.-H., Qiu, J.-H., Schacht, J., 2006. Aspirin attenuates gentamicin-induced hearing loss. New Engl. J. Med. 354, 1856-1857). Based on the finding that salicylate afforded protection in animals, the efficacy of aspirin (acetyl salicylate) was tested in a randomized double-blind placebo-controlled study in patients receiving gentamicin for acute infections. Fourteen of 106 patients (13%) met the criterion of hearing loss in the placebo group while only 3/89 (3%) were affected in the aspirin group (p=0.013). Aspirin did not influence gentamicin serum levels or the course of therapy. These results indicate that therapeutic protection from aminoglycoside ototoxicity may be extrapolated from animal models to the clinic. Furthermore, medications as common as aspirin can significantly attenuate the risk of gentamicin-induced hearing loss.

Diallyl disulfide suppresses growth of HL-60 cell through increasing histone acetylation and p21WAF1 expression in vivo and in vitro.

AIM: To examine the differentiation induction and growth inhibition of HL-60 cells by diallyl disulfide (DADS), and its relationship with the alterations of histone acetylation and p21(WAF1) expression in vitro and in vivo. METHODS: Differentiation was studied by nitroblue tetrazolium (NBT) reduction of HL-60 cell in vitro. HL-60 cells 5x10(6) were injected into the right side of the peritoneal cavity of severe combined immunodeficiency (SCID) mice. When the peritoneal neoplasms were detected, the SCID mice were randomly divided into 3 groups and received an ip injection of vehicle alone (NS), DADS or sodium butyrate (SB). The growth inhibition of peritoneal neoplasms induced by DADS was observed by a growth curve. The cycle distribution of HL-60 cells in SCID mice was monitored by flow cytometry. The expression of acetylated histone H3, H4 and p21(WAF1) were measured by Western blot. RESULTS: After treatment with DADS for 0-72 h, the NBT reduction ability of HL-60 cells increased in a time-dependent manner, compared with no treatment of HL-60 cells. In the HL-60 cells treated with DADS for 24 h, the expression of acetylated histone H3, H4, and p21(WAF1) increased obviously. After treatment with DADS, tumor growth was markedly suppressed. HL-60 cells from mice treated with DADS were blocked in the G1 phase, from 25.4% to 63.4%. The tumors from the mice treated with DADS showed an increase of acetylated histone H3, H4, and p21(WAF1). CONCLUSION: DADS could induce differentiation and inhibit the growth of HL-60 cells through increasing the expression of acetylated histone H3, H4, and p21(WAF1) in vitro and in vivo.

[The results of surgery on HIV carriers with urinary system disease].

OBJECTIVE: To approach characteristics of performing operation on HIV carriers with urinary system diseases. METHODS: To summarize author's experiences of surgery on 41 HIV carriers suffering urinary system diseases abroad from April 1996 to May 2004. RESULTS: The 41 HIV carriers received HAART and were performed with corresponding operations, followed up from 4 to 30 months post-operatively. The 31 carriers have recovered well up to date, while 4 carriers died of AIDS. Among them, 2 patients with penis cancer who received a partial peotomy and a patient with renal tuberculosis receiving left nephrectomy were died of AIDS within 4-8 months after operations whose CD4+ T lymphocyte number was below 0.2 x 10(9)/L. CONCLUSION: Prior to operation, HIV carriers should receive HAART ordinarily to control copy of the virus. The CD4+ T lymphocyte number is important for selecting a proper time for operation and deciding the further after surgery. We also take note to CD4+ T lymphocyte number to monitor progress of the AIDS. For those HIV carriers, endourologic surgery and laparoscopy should be taken so far as possible. Meanwhile, medical stuffs must pay more attention to preventing occupational infection during surgery.

[Human antisense-vascular endothelial growth factor gene therapy for laryngeal tumor by cationic liposome-mediated transfection].

OBJECTIVE: To observe the effect of cationic liposome mediated antisense-vascular endothelial growth factor (VEGF) gene transfection on the growth of laryngeal cancer Hep-2 cells in the nude mice. METHODS: The VEGF-cDNA gene was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) from human laryngeal cancer, and its eukaryotic expression vector pcDNA3-VEGF (-) with antisense-VEGF gene was constructed and identified by PCR and double-enzyme digestion. The pcDNA3-VEGF (-) was transfected into laryngeal cancer Hep-2 cell line by using cationic liposome (LP 2000). Then, the transfected Hep-2 cells were injected into nude mice and the size of tumor from different groups was observed while establishing laryngeal cancer xenografts in nude mice, and then treating the tumor-bearing mice with liposome-plasmid complex, observing the size of tumor from different groups. The expression of VEGF mRNA in different groups was observed by RT-PCR. The transfected cell ultranstructure was observed by transmission electron microscopy. RESULTS: The human VEGF-cDNA was successfully cloned and its eukaryotic expression vector with antisense-VEGF pcDNA3-VEGF (-) was constructed. The antisense-VEGF gene was transfected into Hep-2 cell line by using cationic liposome (LP2000). The size of tumor transfected with pcDNA3-VEGF (-) was significantly smaller than that of control groups. While the size of tumor treated with liposome-pcDNA3-VEGF (-) complex was significantly smaller than that of control groups. Many apoptic tumor cells were observed by transmission electron microscopy and the structure of microvessel was also changed. The expression of VEGF mRNA was evidently weaker than that of the control groups. CONCLUSION: The growth of Hep-2 cells could be inhibited significantly by antisense-VEGF gene transfection.