Altered functional connectivity of the nucleus tractus solitarii in patients with chronic cough after lung surgery: an rs-fMRI study.

BACKGROUND: To explore the altered functional connectivity (FC) of the nucleus tractus solitarii (NTS) in patients with chronic cough after lung surgery using resting-state functional magnetic resonance imaging (rs-fMRI), and the association between abnormal FC and clinical scale scores. METHODS: A total of 22 patients with chronic cough after lung surgery and 22 healthy controls were included. Visual analog scale (VAS), Mandarin Chinese version of the Leicester Cough Questionnaire (LCQ-MC), and Hamilton anxiety rating scale (HAMA) scores were assessed, and rs-fMRI data were collected. The FC analysis was performed using the NTS as the seed point, and FC values with all voxels in the whole brain were calculated. A two-sample t-test was used to compare FC differences between the two groups. The FC values of brain regions with differences were extracted and correlated with clinical scale scores. RESULTS: In comparison to healthy controls, FC values in the NTS and anterior cingulate cortex(ACC) were reduced in patients with chronic cough after lung surgery (GRF correction, p-voxel < 0.005, p-cluster < 0.05) which were positively correlated with LCQ-MC scores (r = 0.534, p = 0.011), but with VAS (r = -0.500, p = 0.018), HAMA (r = -0.713, p < 0.001) scores were negatively correlated. CONCLUSIONS: Reduced FC of the NTS with ACC may be associated with cough hypersensitivity and may contribute to anxiety in patients with chronic cough after lung surgery.

Microstructure changes in whiter matter relate to cognitive impairment in Wilson's disease.

Purpose: Wilson's disease (WD) is a genetic disorder of copper metabolism with pathological copper accumulation in the brain. The purpose of the present study was to evaluate the relationship between the damaged white matter and the impaired cognitive function in WD patients. Materials and methods: Thirty WD adolescents and thirty age- and sex-matched healthy controls (HC) were enrolled. All subjects had received brain MRI, including conventional and diffusion-tensor imaging (DTI) scans. The DTI parameter of fractional anisotropy (FA) was calculated by diffusion kurtosis estimator software. The t test was used to compare the differences between two groups. The correlation between cognitive function and whiter matter disorders were analyzed by linear regression. The results of FA parameter and MD parameter intergroup analysis were both corrected with False Discovery Rate (FDR) simulations by SPSS. Results: WD adolescents showed significantly lower scores of time-based prospective memory (TBPM) and verbal fluency test (VFT) compared with HC. We found significantly higher FA in the right thalamus, right lentiform nucleus, left thalamus, left lentiform nucleus, and brain stem in WD adolescents. Besides, WD adolescents exhibited significantly lower FA in right cerebellum and cingulum and left middle frontal lobe compared with controls (P<0.05). There were significantly negative correlations between FA in bilateral lentiform and thalamus and cognitive impairment in WD adolescents (P<0.05). Conclusion: The whiter matter of WD adolescents was impaired and mainly distributed in subcortical brain regions. The impaired cognitive function was affected by the damaged whiter matter. The present study may be helpful for recognition and understanding of WD.

[Differential Expression of Six wnt Gene Family Members in Echinococcus granulosus Protoscoleces and Adult Worms].

Objective: To investigate the differential mRNA expression and tissue distribution of wnt [wingless-type mouse mammary tumor virus (MMTV) integration site family, wnt] gene members wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B in protoscoleces and adult worms of Echinococcus granulosus. Methods: The mRNA expression of wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B was determined by qRT-PCR. Tissue distribution of wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B in Echinococcus granulosus protoscoleces was determined by the whole-mount in situ hybridization. Results: The qRT-PCR results showed that the mRNA expression levels of wnt1 and wnt2 in the adult worms were 1.49 （P>0.05） and 2.53 folds（P<0.05） of those in the protoscoleces, respectively. The mRNA expression levels of wnt4, wnt5, wnt11A and wnt11B in the protoscoleces were 25.00（P<0.01）, 33.33（P<0.01）, 14.29（P<0.01） and 1.03 folds（P>0.05） of those in the adult worms, respectively. In brief, there was no significant difference of mRNA expression in wnt2 and wnt11B between protoscoleces and adult, but there was a significant difference of mRNA expression in wnt1, wnt4, wnt5 and wnt11A between protoscoleces and adults. Results of the whole-mount in situ hybridization showed that in protoscoleces wnt1 was mainly localized in the epidermal tissue, wnt2 in suckers, wnt4 in suckers and rostellum, wnt5 and wnt11B in suckers and epidermal tissue, and wnt11A in rostellum and hooks. Conclusion: The mRNA expression of wnt2 in adult E. granulosus was higher than that in protoscoleces, and the mRNA expression ofwnt4, wnt5, wnt11A and wnt11B in protoscoleces was higher than that in the adult worms. The six wnt gene family members were all distributed in the forward region of protoscoleces.

Ultrastructure of the nephron in the soft-shelled turtle, Pelodiscus sinensis (Reptilia, Chelonia, Trionychidae).

The structure of the nephron in adult soft-shelled turtles (Pelodiscus sinensis) was studied by light microscopy, transmission and scanning electron microscopy. The kidney contained 5-6 renal lobes. Nephrons of P. sinensis are composed of a renal corpuscle (RC) and of a renal tubule that appears divided morphologically into five distinct segments: neck segment (NS) (This segment is only present in approximately 10% of the nephrons), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting duct (CD). The RCs and most of the convoluted DTs lie in the central zone, while the PTs and the CDs lie in the peripheral zone of the renal lobe. The renal corpuscle is relatively large with especial processes in podocytes and a thick basement membrane. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. The neck segment when presentis short and has a relatively narrow lumen, consisting of cuboidal or squamous cells. There is a well-developed endocytic-lysosomal apparatus in the apical cytoplasm of the PT. The proximal tubule and intermediate segment cells show some differences between male and female. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. Not previously reported in vertebrate kidneys. The IS is the narrowest nephron segment, formed by multiciliated as well as nonciliated cells. In DT cells, basolateral interdigitations and infoldings are particularly well-developed. The CD contains clear cells with numerous secretory granules and dark cells with dense mitochondria and an elaborate Golgi complex. This study was undertaken in order to disclose specific kidney features in P. sinensis that could be related to function. In addition, the ultrastructure of the nephrons in P. sinensis are discussed in relation to other turtles and vertebrates.

Hyperglycaemia exacerbates choroidal neovascularisation in mice via the oxidative stress-induced activation of STAT3 signalling in RPE cells.

Choroidal neovascularisation (CNV) that occurs as a result of age-related macular degeneration (AMD) causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG) antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and vascular endothelial growth factor (VEGF), in mice with CNV. The production of reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC) rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV. Thus, our results reveal a potential strategy for the treatment and prevention of diseases involving CNV.

MSP58 knockdown inhibits the proliferation of esophageal squamous cell carcinoma in vitro and in vivo.

Esophageal carcinoma (EC) is one of the most aggressive cancers with a poor prognosis. Understanding the molecular mechanisms underlying esophageal cancer progression is a high priority for improved EC diagnosis and prognosis. Recently, MSP58 was shown to behave as an oncogene in colorectal carcinomas and gliomas. However, little is known about its function in esophageal carcinomas. We therefore examined the effects of MSP58 knockdown on the growth of esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo in order to gain a better understanding of its potential as a tumor therapeutic target. We employed lentiviral-mediated small hairpin RNA (shRNA) to knock down the expression of MSP58 in the ESCC cell lines Eca-109 and EC9706 and demonstrated inhibition of ESCC cell proliferation and colony formation in vitro. Furthermore, flow cytometry and western blot analyses revealed that MSP58 depletion induced cell cycle arrest by regulating the expression of P21, CDK4 and cyclin D1. Notably, the downregulation of MSP58 significantly inhibited the growth of ESCC xenografts in nude mice. Our results suggest that MSP58 may play an important role in ESCC progression.

[Study on inter-individual variability of cerebral response to acupuncture with fMRI].

OBJECTIVE: Effects of inter-individual variability on fMRI of acupuncture were observed and the possible influencing factors were further analyzed. METHODS: Twenty-six healthy volunteers were selected. And acupuncture was applied at Zusanli (ST 36) on the left side with even manipulation. The same experimental designation and data collecting reference were adopted to collect functional data. Then, the same data processing method was applied for analyzing individual data. Data which did not confirm with data analyzing qualification were rejected. The 26 individual data which met the requirement were taken randomly for 5 times according to the principle of random group division. Five groups named with A, B, C, D and E were thus generated with 11 samples in each. Images were processed with the AFNI software for every group, and the activated brain areas were revealed. RESULTS: Activated areas in the brain were observed in all the 5 groups, and the results vary a lot among different groups. Decreased signals of activated brain areas were observed in group C, while increased signals were seen in group D. Partial increasing and partial decreasing signals appeared in the other 3 groups. Compared with other groups, group D demonstrated totally different activated areas. The rate of difference among different groups is 46.7%-100.0%, and most of the differences were over half of the activated areas. CONCLUSION: Under the pre-requisites of strict control of experimental designation, acupuncture method, data collecting and processing, great differences have been found in the activated areas of the brain. It indicates that obvious individual differences existes in the activated areas of the brain with acupuncture. And the difference may greatly influence the researching result of fMRI as well as conclusions of those results.

[Study on the relative specificity of the heart and lung meridians in brain with fMRI].

OBJECTIVE: To study the central modulation mechanism on the relative specificity of the Heart and Lung Meridians and to provide the experimental evidence for deeply study on correlation between meridian-viscera and brain. METHODS: Ten healthy students in Anhui College of TCM were chosen and a modified block design was adopted. After 32 time points of resting and 32 of rotation needling, then 48 of resting and 32 stimulating, and 16 resting time points, functional imagings were collected at last. All the process would last for 10 min 44 sec. Acupuncture work was finished by one acupuncturist with extensive experience by acupuncture at the left Shenmen (HT 7) or Taiyuan (LU 8) with the disposable sterile stainless steel needle, and uniform reinforcing-reducing method was used with frequency of 1 Hz and depth of 1.0 cm. After the experiment, the sensation of acupuncture and the other feeling or psychic process were inquired and recorded detailedly. These data were analyzed by AFNI software. RESULTS: Acupuncture at Taiyuan (LU 8) could excite the contralateral frontal lobe, apical lobe, cerebral ganglion, VI, VIII areas and inferior semilunar lobule of cerebellum, and restrain bilateral callosal gyrus and homolateral gyrus rectus. Acupuncture at Shenmen (HT 7) could excite the contralateral IV-VIII areas of cerebellum, and homolateral VI, VII areas of cerebellum, and restrain parts of homolateral apical lobe. CONCLUSION: Acupuncture at Shenmen (HT 7) of the Heart Meridian and Taiyuan (LU 8) of the Lung Meridian can excite or restrain different brain areas, indicating that there are relatively specific corresponding brain areas for the Heart Meridian and Lung Meridian.

An immunohistochemical study of S-100 protein in the intestinal tract of Chinese soft-shelled turtle, Pelodiscus sinensis.

The present work describes the distribution of S-100 protein in the intestinal tract of a Chinese soft-shelled turtle specimen (Pelodiscus sinensis). S-100 protein positive cells were located in the intestinal tract, from the proximal small to distal large intestine. S-100 protein positive dendritic cells had irregular shape and were positive in both cytoplasm and nucleus. Most of them were located both lamina propria and submucosa in the small intestine, while few were found in the large intestine. S-100 protein, C-kit positive ICCs and Silver staining glial cells were predominantly observed in three locations: (1) in the interspace between the submucosa and circular muscle layer; (2) in the circular muscle layer; and (3) between the circular and longitudinal muscle layers of the intestine. Fewer were found in the large intestinal lamina propria and submucosa. Three types of positive cells (S-100 protein positive cells, C-kit positive ICCs and Silver staining glial cells) with 1-2 long or 2-3 short processes were distributed as lace-like or surrounding blood vessels in the different locations mentioned above. In the lamina propria, all the positive cells with irregular processes were connected with each other and formed a network. In the submucosa, all the positive cells were found surrounding the blood vessels.

Architecture of the blood-spleen barrier in the soft-shelled turtle, Pelodiseus sinensis.

We investigated the structure of the soft-shelled turtle, Pelodiseus sinensi, spleen and demonstrated that there were several microanatomical peculiarities by light and transmission electron microscopy. In the spleen, the white pulp of the spleen was composed of two compartments, the periarteriolar lymphatic sheath (PALS) and periellipsoidal lymphatic sheath (PELS). No lymph nodules and marginal zones were found. The spleen-blood barrier stood in the PELS and the ellipsoid. The high endothelial lining of penicilliform capillary contained small channels. These channels allowed circulating substances or lymphocytes to enter the ellipsoid. The distal portion of the penicilliform capillaries directly opened to pulp cords. The ellipsoid-associated cell (EAC) was located at the surface of the ellipsoid. Reticular fibers were mainly distributed in ellipsoid and the outer PELS. Both reticular cells and macrophages were distributed in the outer layers of PELS. S-100 protein positive dendritic cells were mainly distributed in out cells layer of the PELS and all over the PALS. Forty minutes after injection, carbon particles of Indian ink were mainly observed in the ellipsoid. Few carbon particles were observed in the outer PELS and fewer carbon particles in the red pulp. These findings suggested that a blood-spleen barrier indeed existed in the soft-turtle, P. sinensi, and it was a complex composed of an ellipsoid (including supporting cells, EAC, and reticular fibers) and the outer compartments of PELS (including dendritic cells, reticular fibers and cells, macrophages).

[Expression and significance of new candidate tumor suppressor gene N-Myc downstream-regulated gene 2 in colorectal cancer].

OBJECTIVE: To determine the expression of new candidate tumor suppressor gene N-Myc downstream-regulated gene 2(Ndrg2) in colorectal cancer with different differentiation, and analyze its clinical significance. METHODS: Specimens of 50 colorectal cancer patients with different differentiation were collected. Immunohistochemistry and Western blot were used to examine the expression of Ndrg2. Colorectal cancer tissue array in large scale was applied to analyze the expression of Ndrg2 and the statistics analysis was performed referring to the patients information of the array. RESULTS: Among 50 cases, Ndrg2 expression level of colorectal cancer was significantly lower in 32 cases as compared to adjacent and normal tissue of the same individual, while Ndrg2 expression of adjacent tissue was significantly lower than that of normal tissue. Ndrg2 protein levels increased from poor-differentiated to well-differentiated carcinomas(P=0.005). CONCLUSIONS: The expression of Ndrg2 in different differentiated colorectal cancer tissues show a significant distinction. Ndrg2 may be involved in the regulation of differentiation in colorectal cancer.

[Relationship of angiotensin-converting enzyme 2 gene polymorphisms and vulnerability to coronary heart disease in patients with type 2 diabetes mellitus].

OBJECTIVE: To investigate the association of angiotensin-converting enzyme 2 (ACE2) gene polymorphisms and coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM). METHODS: This study involved 121 patients with T2DM and 94 with diabetic macroangiopathy. The polymorphisms of G8790A in ACE2 gene was analyzed using PCR-restriction fragment length polymorphism analysis in these patients, and the clinical, biochemical and echocardiographic data were also analyzed. RESULTS: No obvious difference was found in the genotyping data between the two groups. Among the male patients with diabetic macroangiopathy, the interventricular septal end-diastolic thickness (IVSTd) were significantly greater in patients of GG genotypes of ACE2 gene G8790A than in those of AA genotypes (P<0.01), and the left ventricular mass (LVMI) and urine protein were also significantly higher in GG genotypes (P<0.05). No similar results were found the uncomplicated diabetic group or the female diabetic patients with CAD. CONCLUSION: The ACE2 gene G8790A polymorphism plays a role in the pathogenesis of CAD in patients with type 2 diabetes, suggesting that ACE2 genotyping is helpful to screen the susceptible patients.

[Effect of enalapril on diabetic rat myocardial ultrastructure].

OBJECTIVE: To observe the changes in the myocardial ultrastructure of diabetic rats and the effect of enalapril treatment. METHODS: Male Wistar rats were divided into 3 groups, namely the control group, diabetic group and enalapril intervention group. Diabetes was induced with peritoneal injection of streptozotocin in the latter 2 groups, and in enalapril group, the rats were treated with enalapril at the daily oral dose of 2 mg/kg for 1, 3 and 5 months after streptozotocin injection. Histological analysis of the left ventricular tissue was performed with transmission electron microscope 1, 3, and 5 months after establishment of diabetes. RESULTS: Onset of myocardial damages was observed 1 month after the development of diabetes in the rats with gradual time-dependent exacerbation. Enalapril treatment could partially reverse the myocardial destruction in the diabetic rats. CONCLUSION: Enalapril intervention may improve the ultrastructural pathology of the myocardium in diabetic rats, which is suggestive of the action mechanisms of angiotensin-converting enzyme inhibitors in myocardium preservation.

[Effects of proadrenomedullin N-terminal 20 peptide on angiotensin II-induced NO production in rat cardiac fibroblasts].

OBJECTIVE: To explore the effects of proadrenomedullin N-terminal 20 peptide (PAMP) on nitric oxide (NO) production in rat cardiac fibroblasts (CFs) induced by angiotensin II (AngII) stimulation. METHODS: Neonatal SD rat CFs isolated by trypsin digestion were cultured and stimulated with PAMP, AngII or their combination, and NO production in the CFs in response to the treatments was measured by nitric acid reductase method. RESULTS: NO levels in the cell culture treated with 1x10(-9), 1x10(-8), 1x10(-7), and 1x10(-6) micromol/L AngII were 73.88-/+2.23, 64.34-/+3.02, 54.12-/+2.82, and 40.21-/+1.45 micromol/L, respectively, showing significant differences between the groups (P<0.01), whereas treatment of the cells with 1x10(-9), 1x10(-8), 1x10(-7), and 1x10(-6) micromol/L PAMP did not result in significant variation in NO production (74.40-/+3.42, 74.91-/+2.66, 75.77-/+3.31, and 74.23-/+2.43 micromol/L, respectively) in comparison with that of the blank control group (74.57-/+2.49 micromol/L, P>0.05). Combined treatments with 1x10(-7) micromol/L AngII and PAMP at 1x10(-9), 1x10(-8), 1x10(-7), and 1x10(-6) micromol/L PAMP caused significant increment of NO production (66.15-/+2.95, 80.58-/+3.77, 88.67-/+1.46, and 96.22-/+2.96 micromol/L, respectively, P<0.01) in a PAMP dose-dependent manner, suggesting the abolishment of AngII-induced enhancement of NO production in the CFs by PAMP. CONCLUSION: PAMP increases NO production in the CFs in the presence of AngII but it does not induce significant changes in NO production when used alone.

[Effect of urotensin II on secretion of adrenomedullin from human vascular endothelial cells].

OBJECTIVE: To study the effect of human urotensin II (HU II) on secretion of adrenomedullin (ADM) from human vascular endothelial cells (HVEC) and its mechanism. METHODS: In cultured HVEC, different concentrations of HUII were used to stimulate the ADM secretion from HVEC, and the inhibitors of different signal transduction pathway were used to investigate their effects on ADM secretion. The contents of ADM in medium were determined by radio immunoassay. RESULTS: HUII stimulated secretion of ADM from HVEC in a time-dependent and concentration-dependent manner. The contents of ADM in the experiment groups were changed compared with that in control group (P < 0.05). The increase of ADM could be inhibited by inhibitor of extracellular signal-regulated protein kinase (PD(98059)), inhibitor of P38 kinase (SB(202190)), inhibitor of calmodulin (W(7)) and inhibitor of Ca(2+) (nicardipine) (P < 0.05). The inhibition ratio in those groups was 68%, 78%, 24% and 25% respectively. But the inhibitor of Calcineurin (CaN) and inhibitor of protein kinase C (H(7)) had no influence on the secretion of ADM from HVEC (P > 0.05). CONCLUSION: The stimulated effect of HUII on the ADM secretion from HVEC may be mediated by Ca(2+), ERKs, CaM-PK and P38 signal transduction pathways.

[Dynamic changes in plasma adrenomedullin levels and effect of enalapril intervention in diabetic rats].

OBJECTIVE: To observe the dynamic changes of plasma adrenomedullin (ADM) levels and the effect of enalapril intervention in diabetic rats. METHODS: Fifty-two wistar rats were grouped into 1- and 3-month groups (7 each), both including a control and a streptozotocin-induced diabetic group. The 5-month group was divided into control, diabetic and enalapril-treatment diabetic groups (8 each, the last group receiving oral enalapril treatment at the daily dose of 2 mg/kg from the first to the fifth month after diabetes induction). Blood samples were collected from the heart of the rats at the end of 1, 3 and 5 months for determination of plasma ADM concentrations radioimmunoassay. RESULTS: Plasma ADM levels were significantly higher in diabetic rats than in the corresponding control rats in 1- and 3-month groups. ADM levels in the diabetic rats of 5-month group were significantly decreased in comparison with that of 1- and 3-month groups. In 5-month group, plasma ADM levels of enalapril-treated diabetic rats elevated significantly in comparison with that in the control rats and the diabetic rats without enalapril treatment. CONCLUSION: ADM may play an important role in the pathophysiology of diabetes.