Effect of electroacupuncture at Neiguan (PC6) at different time points on myocardial ischemia reperfusion arrhythmia in rats.

OBJECTIVE: To observe the effects of electroacupuncture at Neiguan (PC6) at different time points on reperfusion arrhythmia (RA) after myocardial ischemia and reperfusion in rats, and to investigate the correlation of this protective effect with nerve growth factor (NGF), tyrosine kinase A (TrkA), tyrosine hydroxylase (TH), and norepinephrine (NE). METHODS：A total of 72 Sprague-Dawley male rats were randomly divided into six groups (n = 12 rats/group): normal group (Norm), sham operation group (Sham), ischemia reperfusion group (I/R), pre-ischemic electroacupuncture group (EAI), pre-reperfusion electroacupuncture group (EAII), post-reperfusion electroacupuncture group (EAIII). The myocardial ischemia-reperfusion injury (MIRI) model was induced by occlusion of left anterior descending coronary artery for 20 min followed by reperfusion for 40 min in rats. With no intervention in the Norm group and only threading without ligation in the Sham group. Electroacupuncture pre-treatment at 20 min/d for 7 d before ligation in the EAⅠ group, 20 min of electroacupuncture before reperfusion in the EAII group and 20 min of electroacupuncture after reperfusion in the EAIII group. The electrocardiogram (ECG) of each group was recorded throughout the whole process, and the success of the MIRI model was determined based on the changs of J-point and T-wave in the ECG. The arrhythmia score was used to record premature ventricular contractions, ventricular tachycardia and ventricular fibrillation during the reperfusion period to assess the reperfusion induced arrhythmias. The expression levels of NGF, TrkA, TH protein were measured by Western blot. Moreover, the expression levels of plasma and myocardial NE levels were detected by enzyme linked immunosorbent assay. RESULTS: The differences between Norm group and Sham group were not statistically significant in all indexes. Arrhythmia score, myocardial NGF, TrkA, TH, and NE expression were significantly higher in the I/R group compared with the Sham group. Arrhythmia score, myocardial NGF, TrkA, TH, and NE expression were significantly lower in each EA group compared with the I/R group. CONCLUSION: Electroacupuncture at Neiguan (PC6) at different time points can reduce the incidence and severity of reperfusion arrhythmias in rats. This protective effect is related to electroacupuncture regulating NGF, TrkA, TH, NE expression and reducing sympathetic hyperactivation.

Coordinated Regulation of Myelination by Growth Factor and Amino-acid Signaling Pathways / 神经科学通报·英文版

Myelin-forming oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are essential for structural and functional homeostasis of nervous tissue. Albeit with certain similarities, the regulation of CNS and PNS myelination is executed differently. Recent advances highlight the coordinated regulation of oligodendrocyte myelination by amino-acid sensing and growth factor signaling pathways. In this review, we discuss novel insights into the understanding of differential regulation of oligodendrocyte and Schwann cell biology in CNS and PNS myelination, with particular focus on the roles of growth factor-stimulated RHEB-mTORC1 and GATOR2-mediated amino-acid sensing/signaling pathways. We also discuss recent progress on the metabolic regulation of oligodendrocytes and Schwann cells and the impact of their dysfunction on neuronal function and disease.

Effect of electroacupuncture on inflammatory signal expression in local tissues of rats with chronic pelvic pain syndrome based on purinergic 2X7 receptor/NOD-like receptor pyrin domain-containing 3 signal pathway.

OBJECTIVES: To study the expression of inflammatory signal in local prostate tissue of chronic pelvic pain syndrome (CPPS) rats by electroacupuncture (EA) of Guanyuan (CV4), Zhongji (CV3), Huiyang (BL35) and Sanyinjiao (SP6), and to explore the possible mechanism of anti-inflammatory and analgesic effects of EA. METHODS：A total of 36 Sprague-Dawley male rats were randomly divided into three groups: control, model and EA (n=12 rats/group). The CPPS model was made by injection of CFA into ventral lobes of the prostate (0.1 mL). Electric acupuncture apparatus was applied to stimulate Guanyuan (CV4), Zhongji (CV3), bilateral Huiyang (BL35) and Sanyinjiao (SP6) acupoints in EA group. The general condition of rats was observed and the prostate index (PI) was calculated. The thermal pain threshold was collected after each therapeutic course. Histopathological changes of the prostate tissue were examined by hematoxylin-eosin staining method. The expression levels of tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) in prostatic homogenates were measured by enzyme linked immunosorbent assay (ELISA). Moreover, the expression levels of purinergic 2X7 receptor (P2X7R), NOD-like receptor pyrin domain-containing 3 (NLRP3), caspase-1 and interleukin-18 (IL-18) mRNA were quantified by quantitative real-time polymerase chain reaction. RESULTS: Compared with control group, the PI of rats increased, and the thermal pain threshold decreased significantly in model group. The morphological structure of prostate tissues of rats in model group was severely damaged with a large number of inflammatory cells infiltration. Additionally, the levels of TNF-α, IL-1ß and PGE2 were higher, and the expressions of P2X7R, NLRP3, caspase-1 and IL-18 mRNA were higher than those in control group. After EA treatment, the PI was significantly decreased, the thermal pain threshold was significantly increased, and the tissue damage was significantly improved. The expressions of inflammatory cytokines were lower in EA group, and expression of P2X7R/NLRP3 pathway was down-regulated. CONCLUSION: The effect of EA at Guanyuan (CV4), Zhongji (CV3), Huiyang (BL35) and Sanyinjiao (SP6) can improve inflammation and pain symptoms of CPPS rats induced by Complete Freund's adjuvant (CFA). This suggests that EA at Guanyuan (CV4), Zhongji (CV3), Huiyang (BL35) and Sanyinjiao (SP6) can produce anti-inflammatory analgesia effect by preventing the activation of P2X7R/NLRP3 signal pathway, inhibit the release of inflammatory cytokines in CPPS rats, which may provide a putative novel target for the treatment of CPPS.

Postpolypectomy fever in patients with serious infection: a report of two cases.

BACKGROUND: Postpolypectomy fever (PPF) is a rare complication in patients after colonoscopy. Because of the absence of evidence of microperforation and abdominal tenderness, patients with PPF usually present mild clinical symptoms with a good prognosis. CASE PRESENTATION: In this study, all patients who underwent colonoscopic examination in our hospital between January 2019 and December 2019 were enrolled. Of these, two patients developed PPF after polypectomy, exhibiting serious infection without definitive fever foci. One patient experienced rapidly aggravated type 1 respiratory failure and abnormal hepatic function, which were attributed to colonoscopy-associated infection. After active antibiotic therapy, both patients were discharged without any complications. CONCLUSIONS: In summary, our study provides novel insights into patients with PPF who develop serious infections with life-threatening complications.

Relationship between caspase recruitment domain protein 9 gene polymorphism and clinical efficacy of somatostatin and acute pancreatitis / 中国综合临床

Objective:To investigate the relationship between caspase recruitment domain protein 9 (CARD9) gene polymorphism and acute pancreatitis (AP) and the clinical efficacy of somatostatin.Methods:A total of 86 patients with AP treated in Shanghai Songjiang District Central Hospital from June 2019 to may 2020 were selected as the research object, and 81 healthy volunteers were selected as the control group for a prospective cohort study. The nucleotide database of National Center for Biotechnology Information (NCBI) was consulted to screen 10 common single nucleotide polymorphisms of CARD9.The single nucleotide polymorphism of CARD9 was detected by SNapShot micro sequencing. All patients with AP were treated with somatostatin. The relationship between CARD9 single nucleotide polymorphism and clinical symptoms and auxiliary examination indexes was observed.The measurement data of normal distribution were compared by independent sample t-test. The measurement data of non normal distribution are represented by M (Q1, Q3), and the rank sum test is used for comparison between groups. The comparison of counting data between groups was adopted χ 2 inspection. Results:The frequency of CARD9 rs10870077 C>G SNP in patients of AP group was significantly higher than that in healthy controls ( OR=1.934, 95% CI=1.011-3.700, P=0.046). Compared with CC genotype, the disappearance time of abdominal pain and abdominal distension in the somatostatin treatment group of CARD9 rs10870077 C>G moderate and severe AP patients was significantly longer ((5.64±2.06) d and (3.76±1.23) d, t=2.98, P=0.006), and the average hospital stay in the somatostatin treatment group of CARD9 rs10870077 C>G severe AP patients was increased by ((13.25±5.31) d and (9.00±3.68) d, t=1.51, P=0.170). Conclusion:CARD9 rs10870077 C>G is a predisposing factor for AP, which is related to the individual differences in the clinical efficacy of somatostatin in severe AP.

Correlation between caspase recruitment domain protein 9 gene polymorphism and susceptibility to sepsis related liver injury / 中国综合临床

Objective:To investigate the relationship between caspase recruitment domain protein 9 (CARD9) gene polymorphism and the risk of sepsis related liver injury.Methods:A case-control study was conducted to recruit 122 patients with sepsis in intensive care unit (ICU). The septic patients with liver injury were divided into the liver injury group ( n=66), and the patients without liver injury were set as the control group ( n=60). TaqMan genotyping technique was performed to detected the six single nucleotide polymorphisms of CARD9 gene in peripheral blood, and the baseline data of the two groups were observed. The differences of genotype distribution and allele frequency between the two groups were compared, and the odds ratios ( OR) were calculated. Kolmogorov-Smirnov test was used for normal distribution test, the measurement data that do not conform to the normal distribution are expressed in M ( Q1, Q3), and the Mann Whitney U test was used for the comparison between the two groups. The comparison of counting data between groups was adopted χ 2 inspection and Fisher exact probability method. χ 2 test is used to determine whether the genotype distribution conforms to hardy Weinberg equilibrium test. Results:Compared with the control group, the CC genotype distribution of rs10781500 locus of CARD9 gene in liver injury group was significantly increased (19 vs. 36, χ 2=6.87, P=0.033), and sepsis patients with C allele are more likely to induce liver injury ( OR=1.375, 95% CI 1.024-1.846, P=0.023). Patients with CC genotype have a higher risk of liver injury ( OR=2.696, 95% CI 1.238-5.869, P=0.012). In the liver injury group, the alanine aminotransferase and SOFA score in CC genotype at re10781500 locus were significantly higher than those in non-CC genotype (310 (213, 648) U/L vs. 187 (114, 304) U/L, Z=3.32, P=0.001 and 9 (6, 14) points vs. 7 (5, 9) points, Z=2.49, P=0.013). Conclusion:The CC genotype at rs10781500 locus of CARD9 gene was associated with sepsis-induced liver injury, and the C allele may be a susceptibility gene.

The role of caspase recruitment domain protein 9 in pancreatic acinar-to-ductal metaplasia / 中华胰腺病杂志

Objective:To investigate the role of caspase recruitment domain protein 9(card9) from macrophage in pancreatic acinar-to-ductal metaplasia.Methods:Card9 siRNA1, card9 siRNA2 and card9 siRNA3 were constructed; fluorescence microscopy was used to investigate the fluorescence intensity of macrophages, and real-time quantitative PCR method was performed to detect the expressed level of card9 mRNA to obtain the best transfection rate. 100 μg/ml β glucan was added into 5×10 5 macrophages in vitro culture for 12 or 24 hours, which were divided into positive group (macrophages), β glucan-stimulated positive group (β dextran+ macrophage), negative group (card9 -/- macrophage) and β glucan-stimulated negative group (β dextran+ card9 -/- macrophages). Western blotting was applied to determine the protein level of card9 in macrophages. Then, 1×10 5 macrophages and 1×10 5 pancreatic acinar cells were co-cultured in upper and lower transwell chamber in vitro for 120 hours, which were divided into positive group (macrophages+ acinar cells), 100 μg/ml and 500 μg/ml β glucan-stimulated positive group, negative group (card9 -/- macrophage+ acinar cell), 100 μg/ml and 500 μg/ml β glucan-stimulated negative group. Pancreatic acinar cells in the lower chamber were collected and immunofluorescence was applied to assay the duct metaplasia marker CK19 protein expression. Results:At 24 hours of transfection using siRNA, the intracellular fluorescence intensity in macrophages reached a peak. Card9 siRNA at the concentration of 200 nmol/l showed the highest interference efficiency. Card9 protein in positive group, β glucan-stimulated positive group, negative group, and β glucan-stimulated negative group were 0.81±0.05, 1.46±0.05, 0.42±0.06 and 0.46±0.06, respectively; card9 expression in β glucan-stimulated positive group was obviously higher than that in positive cell group, and the difference was statistically significant ( P<0.05). Finally, after 100 or 500 μg/ml β glucan stimulation, the green fluorescence in pancreatic acinar cells increased significantly compared with positive group, exhibiting β glucan concentration dependence. Conversely, CK19 protein in negative group and 100 and 500 μg/ml β glucan-stimulated negative group was obviously decreased compared with positive group. Conclusions:The expression level of card9 in macrophages can induce acinar-to-ductal metaplasia, indicating that card9 may mediate in the pathogenesis of pancreatic cancer.

Role of telomerase reverse transcriptase on cell apoptosis of neutrophils in rats with acute necrotizing pancreatitis / 中华胰腺病杂志

Objective:To investigate the effects of telomerase reverse transcriptase (TERT) on neutrophils apoptosis in rats with acute necrotizing pancreatitis (ANP).Methods:Twenty-four Sprague Dawley (SD) rats were randomly divided into three groups including control group, ANP (3h、6h) group and TERT inhibitor(BIBR1532)group using random number method with 6 in each group. ANP rats were induced by retrograde injection of 5% sodium taurocholate into the pancreaticcobiliary duct. After 3 and 6 hours, the fresh neutrophils were collected and isolated from peripheral blood of ANP rats. Rats were intraperitoneally injected with 2 mg/kg BIBR1532. After ANP modeling for 3 h, rats were killed and peripheral neutrophils were collected. Subsequently, the expression of TERT mRNA in neutrophils was tested by real-time quantitative PCR; the protein levels of TERT, BCL-xL and Bax were determined by Western blotting; neutrophil apoptosis was detected by flow cytometry; TNF-α and IL-6 were assayed by Elisa; the rat pancreatic tissue was pathologically examined.Results:In neutrophils from control group, ANP 3 h group, ANP 6 h group and BIBR1532 group, TERT mRNA was 1.03±0.26, 3.31±1.07, 5.21±0.78 and 1.95±0.49; TERT protein expression was 0.09±0.03, 0.43±0.12, 0.58±0.11 and 0.22±0.07; Bcl-xL protein expression was 0.19±0.05, 0.50±0.07, 0.85±0.04 and 0.40±0.11; Bax protein expression was 0.29±0.08, 0.23±0.03, 0.17±0.02 and 0.43±0.12; apoptosis rate was 10.03±0.74%, 7.99±0.27%, 6.65±0.36% and 22.98±2.86%. TERT mRNA and protein expression and Bcl-xL protein expression in ANP 3 h and 6 h group were higher than those in control group, but Bax preotein expression and apoptosis rate were lower than those in control group; TERT mRNA and protein expression and Bcl-xL protein expression in BIBR1532 group were lower than those in ANP 3 h group, but Bax protein expression and apoptosis rate in BIBR1532 group were higher than those in ANP 3 h group; and all the differences were statistically significant (all P value <0.05). The level of TNF-α was [(96.67±27.12)ng/L, (382.30±46.33)ng/L and (206.88±36.42)ng/L], IL-6 was [(43.34±14.50)ng/L, (134.21±16.13)ng/L and (88.06±13.05)ng/L in control, ANP 3 h and BIBR1532 group; those in ANP 3 h group were all higher than those in control group, but those in BIBR1532 group were lower than those in ANP 3 h group; all the differences were statistically significant (all P value <0.05). Compared with ANP group, the morphology of pancreatic tissue was obviously alleviated in BIBER1532 group. Conclusions:TERT expression is obviously increased in peripheral neutrophils in ANP rats, and apoptosis rate of neutrophils from ANP rats is greatly increased after TERT inhibitor treatment, demonstrating that TERT could inhibit the apoptosis of peripheral neutrophil in ANP rats.

Association of caspase recruitment domain protein 9 gene polymorphism with the risk of acute pancreatitis / 中华胰腺病杂志

Objective:To evaluate the correlation between Card 9 gene polymorphism and acute pancreatitis(AP).Methods:70 AP patients and 70 healthy subjects from Shanghai Songjiang District Central Hospital from June 2019 to February 2020 were enrolled. TaqMan probe method was used to assay genotype distributions of the Card 9 polymorphisms rs10870077, rs4077515, rs10781499, rs141992399, rs139265120. Real-time quantitative PCR was used to determine the level of Card 9 mRNA, electrochemiluminescence immunoassay was applied to assay IL-6 and procalcitonin, and nephelometry was performed to measure the C-reactive protein(CRP).Results:Compared with the control group, the genotype and allele frequency of Card 9 gene rs10870077 C>G were significantly elevated in AP patients with statistically significant difference (31.4% vs 50.0%, P<0.05). There was no statistically significant difference on the allele frequency of Card 9 rs4077515AG and rs10781499AG, especially on rs141992399 C>G and rs139265120 A>G. C>Gpolymorphism in Card 9 rs10870077 resulted in an obvious increase of serum Card 9 mRNA expression in AP patients from 3.90±1.96 to 6.20±2.82, and the difference was statistically significant ( P<0.05), but there was no statistically significant difference on the Card 9 mRNA between AP patients with Card 9 rs4077515AG and rs10781499AG and the controls. IL level in AP patients with Card 9 rs10870077CG was greatly higher than that in those with Card 9 rs10870077 CC and GG [(614.7±1531.8 ng/L vs (372.5±1127.9)and (385.5±598.7)ng/L]. But compared with GG genotype, CRP level was obviously decreased [(34.84±50.64)mg/L vs (55.30±87.02)mg/L], and the procalcitonin was obviously increased [(1.98±4.70)μg/L vs (0.77±1.12)μg/L], and all the differences were statistically significant (all P value<0.05). Conclusions:Card 9 rs10870077 C>G mutation could upregulate the expression of Card 9 mRNA and increase IL-6 level, which may be a high risk for the occurrence of AP patients.

Effects of inhibiting peritoneal macrophage caspase recruitment domain-containing protein 9 on inflammatory reaction of severe acute pancreatitis in rats / 中华消化杂志

Objective To investigate the effects of caspase recruitment domain-containing protein 9 (CARD9)expression in peritoneal macrophages on severe acute pancreatitis(SAP)in rats and its mechanism.Methods A total of 60 male Sprague Dawley rats were divided into control group(n=6), SAP group(n=18),small interfering RNA(siRNA)control group(n=18)and siRNA CARD9 group (n=18).SAP rat models were established.At three,six and twelve hours after the models were established,ascites was collected,peritoneum was lavaged and peritoneal macrophages were isolated and cultured.The expressions of CARD9,nuclear factor-kappaB(NF-κB),p38 mitogen-activated protein kinase(p38MA PK)at mRNA level in peritoneal macrophages was measured by real-time polymerase chain reaction(RT-PCR).The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6 in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA).LSD or Tamhane′s T2 methods were performed for statistical analysis.Results At three,six and twelve hours after the models were established,CA RD9 mRNA levels of peritoneal macrophages in SAP group were 1.63 ± 0.05,1.68 ± 0.24 and 2.61 ± 0.02,respectively,which were all higher than that of control group(1.01 ± 0.23),and the differences were statistically significant(t=25.97,6.86 and 131.59;all P<0.05);the levels of CA RD9 mRNA of siRNA CARD9 group were 1.45 ± 0.02,1.24 ± 0.03 and 1.63 ± 0.03,respectively,which were lower than that of SAP group at the same time points,and the differences were statistically significant(t=-7.81,-4.46 and -62.13;all P< 0.05).At three,six and twelve hours after the models were established,the mRNA levels of NF-κB and p38MA PK of peritoneal macrophages of rats in SAP group were 1.51 ± 0.08,1.81 ± 0.10,2.30 ± 0.05 and 1.37 ± 0.13,1.69 ± 0.18,2.42 ± 0.23,respectively, which were higher than those of control group(1.00 ± 0.01,1.03 ± 0.08),and the differences were statistically significant(tNF-κB=15.10,19.95 and 60.36;tp38MAPK=5.37,8.34 and 14.11;all P<0.05);the levels of N F-κB mRNA in siRNA CARD9 group were 1.38 ± 0.05,1.57 ± 0.06 and 1.76 ± 0.09, respectively,which were lower than that of SAP group at the same time points,and the differences were statistically significant(t= -3.32,-5.07 and -12.70;all P<0.05).At six and twelve hours after the models were established,the p38MA PK mRNA levels of siRNA CARD9 group were 1.50 ± 0.10 and 2.00 ± 0.09,respectively,which were lower than that of SAP group,and the differences were statistically significantly(t= -2.30 and -4.17,both P< 0.05).At three,six and twelve hours after the models were established,the levels of TNF-α,IL-1β and IL-6 in peripheral blood of SAP group were(53.49 ± 21.64)pg/mL,(108.62 ± 22.76)pg/mL and(139.00 ± 15.35)pg/mL;(43.86 ± 18.30)pg/mL, (87.51 ± 17.10)pg/mL and(117.27 ± 14.57)pg/mL;(78.38 ± 32.70)pg/mL,(156.39 ± 30.56)pg/mL and(209.56 ± 26.09)pg/mL,respectively,which were higher than those of control group((2.79 ± 1.17),(7.13 ± 4.52),(12.73 ± 8.08)pg/mL),and the differences were statistically significant(tTNF-α=5.73,11.37 and 21.69;tIL-1β=4.77,11.13 and 17.68;tIL-6=4.77,11.32 and 17.68;all P<0.05).At six and twelve hours after the models were established,the levels of TNF-α,IL-1β and IL-6 of siRNA CARD9 group were(75.73 ± 16.93)pg/mL,(108.23 ± 14.02)pg/mL;(63.05 ± 11.98)pg/mL, (91.56 ± 14.28)pg/mL and(112.67 ± 21.40)pg/mL,(163.62 ± 25.51)pg/mL,respectively,which were lower than those of SAP group,and the differences were statistically significant(tTNF-α= -2.84,-3.63;tIL-1β= -2.88,-3.09;tIL-6= -2.88,-3.09;all P< 0.05).Conclusions There are CARD9-related NF-κB and p38MAPK pathways in peritoneal macrophages of SAP rats.Intervention of the expression of CARD9 in peritoneal macrophages,especially at early stage of SAP may relieve the inflammation reaction and pancreatic injury,which may provide a new method for SAP treatment.

Association study between a genetic polymorphism in cytochrome P450 19 gene rs10046 and colon and rectal cancer / 中国医师杂志

Objective To investigate the relationship between the polymorphism of cytochrome P450 19 (CYP19) gene rs10046 and the risk of colon and rectal cancer.Methods Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to analyze gene polymorphism in CYP 19 gene rs10046 in 198 cases of colon and rectal cancer patients (case group) and 309 cases of healthy controls (control group).The genotype frequency and relative risk of CYP19 gene rs10046 between the two groups were compared and the relationship with the clinicopathological features of colorectal cancer was analyzed.Results In case group,the prevalence rates of CYP19 rs10046 genotypes C/C,C/T and T/T were 28.3％,44.4％ and 27.3％,respectively,and 17.2％,51.8％ and 31.1％ in the control group,respectively,with statistical significant difference (P ＜ 0.05).Compared with wild-type C/C,the susceptibility of colorectal cancer with the genotypes of C/T and T/T was decreased by 0.521 (95％ CI:0.330-0.822)and 0.532 (95 ％ CI:0.322-0.880) respectively.Moveover,in the non-smoking group,the risk of colorectal cancer with genotype T/T or C/T was decreased by 0.409 (95％ CI:0.210-0.798) compared with genotype C/C.The interaction was not exist in smoking group.Conclusions The polymorphism of CYP19 gene rs10046 is related to the susceptibility of colon and rectal cancer.The T/T,C/T genotype of CYP19 rs10046 decrease the risk of colon and rectal cancer,and which might be the protective factor of colon and rectal cancer.

Effect of Agonist and Inhibitor of PI3K/AKT on Inflammatory Response in Macrophages / 胃肠病学

Phosphoinositide 3-kinase/serine-threonine kinase (PI3K/AKT)has been found playing an important role in the pathogenesis of severe acute pancreatitis (SAP)in recent years,but the underlying mechanism has not been clarified.Aims:To investigate the role of PI3K/AKT in regulating the inflammatory response in SAP by evaluating the effect of insulin-like growth factor-Ⅰ (IGF-Ⅰ)and wortmannin,the agonist and inhibitor of PI3K/AKT on Toll-like receptor 4 (TLR4)signaling pathway in macrophage cell line RAW264.7.Methods:RAW264.7 cells were treated with different concentrations of lipopolysaccharide (LPS ), IGF-Ⅰ and wortmannin, respectively, and cell viability was determined by CCK-8 assay.RAW264.7 cells were divided into blank control group (no treatment),LPS group (LPS 1 μg/mL),IGF-Ⅰ group (IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL),wortmannin group (wortmannin 100 nmol/L +LPS 1 μg/mL)and IGF-Ⅰ +wortmannin group (wortmannin 100 nmol/L +IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL).Protein expressions of tumor necrosis factor-α(TNF-α)and interleukin-6 (IL-6)were detected by ELISA;mRNA expressions of TLR4,myeloid differentiation factor 88 (MyD88),AKT,PI3K,p38 mitogen-activated protein kinase (p38MAPK)and nuclear factor-κB (NF-κB)were determined by real-time PCR.Results:After treated with LPS,IGF-Ⅰand wortmannin, respectively,no differences in cell viability of RAW264.7 cells were found between different concentrations groups (P>0.05).Protein expressions of TNF-αand IL-6 in LPS,IGF-Ⅰ,wortmannin and IGF-Ⅰ +wortmannin groups were significantly higher than those in blank control group (P<0.05 ).Protein expressions of TNF-αand IL-6 in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups (P<0.05),and those in IGF-Ⅰ+wortmannin group were significantly lower than those in IGF-Ⅰ group (P<0.05).In LPS group,mRNA expressions of AKT and PI3K as well as TLR4 and its downstream molecules MyD88,p38MAPK and NF-κB were significantly higher than those in blank control group (P <0.05 ).Expressions of all above-mentioned mRNAs in IGF-Ⅰ group were further increased and significantly higher than those in LPS group (P<0.05).Expressions of all above-mentioned mRNAs in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups (P<0.05 ),and those in IGF-Ⅰ+wortmannin group were significantly higher than those in wortmannin group (P<0.05),but significantly lower than those in IGF-Ⅰ group (P<0.05).Conclusions:PI3K/AKT might regulate TLR4 signaling pathway and its downstream molecules in macrophages, thereby affects the expressions of inflammatory cytokines and being involved in the pathogenesis of inflammatory response in SAP.

Emerging Role of microRNA in Neuropathic Pain.

BACKGROUND: Neuropathic pain is an incurable disease which is defined as a chronic pain caused by a disease or lesion of the nervous systems. Current treatments can provide a long-lasting pain relief only in a very limited number of patients with neuropathic pain. MicroRNA can regulate multiple genes and pathways involved in human diseases. This review focuses on: a) Molecular mechanisms of microRNA biogenesis. b) Targeting, modifications, and delivery of microRNAs. c) Aberrant expression of microRNAs and their potential therapeutic targets in neuropathic pain. d) Potential challenges of microRNA therapy in clinical practice, such as off-target effects, toxicity, delivery hurdles, and target prediction. RESULTS: This review introduces: 1. Canonical/non-canonical pathway of microRNA biogenesis. 2. Viral/non-viral vectors transporting microRNAs into target cells. 3. MicroRNA mimics/inhibitors targeting strategies. 4. Aberrantly expressed microRNAs in different animal neuropathic pain models and their links to underlying mechanisms such as inflammation and ion channel expression. 5. Potential challenges of microRNAs therapy such as off-target effect, pseudogenes, liver toxicity, delivery hurdles and target prediction. CONCLUSION: Although using microRNAs to target neuropathic pain seem promising, their off-target/toxic effects and delivery hurdles still need to be surmounted.

Differentially Expressed miRNAs in Peripheral Blood of Patients with Recurrent Colorectal Adenoma / 胃肠病学

Background:MicroRNAs ( miRNAs ) is a class of small non-coding RNAs that regulate gene expression at posttranscriptional level. They are considered to play a critical role in the tumorigenesis and development of various tumors. Aims:To identify the recurrence-specific miRNAs in peripheral blood of patients with recurrent colorectal adenoma by miRNA array for providing sensitive and specific molecular markers for recurrence prediction. Methods:Fifty colorectal adenoma patients who had undergone polypectomy by colonoscopy were re-examined from Aug. 2012 to Sep. 2013. Four recurrent and four non-recurrent patients were selected and miRNA array was used to detect the expression profiles of miRNAs in peripheral blood samples. Differentially expressed miRNAs were identified between recurrent and non-recurrent patients. Real-time PCR was applied to verify the differentially expressed miRNAs identified by microarray. Results:Seven up-regulatedmiRNAs(hp hsa-miR-548ai,hsa-miR-4446-3p,hsa-miR-139-3p,hsa-miR-27a-5p,hsa-miR-10a,hsa-miR-23a and hsa-miR-486-3p)and 4 down-regulated miRNAs( hsa-miR-124,hsa-miR-3613-5p,hsa-miR-495 and hsa-miR-485-5p)were identified by microarray between recurrent and non-recurrent patients. Among them,seven miRNAs were validated by real-time PCR as consistent with microarray. Conclusions:Some differentially expressed miRNAs may be involved in the recurrence of colorectal adenoma,which gives clues for further investigation on recurrence predictors.

Mechanism on the role of caspase recruitment domain-containing protein 9 in the early stage of acute pancreatitis / 中华消化杂志

Objective To investigate the mechanism of caspase recruitment domain‐containing protein 9 (CARD9) in the early stage of acute pancreatitis(AP) .Methods Peripheral blood mononuclear cells (PBMC ) of 49 AP patients (33 mild acute pancreatitis (MAP ) patients and 16 severe acute pancreatitis (SAP) patients) were collected on the Day 1st ,3rd and 5th of hospitalization .Twenty healthy volunteers were enrolled in control group .The expression level of CARD9 ,B‐cell lymphoma(Bcl)‐10 ,p38 mitogen‐activated protein kinase (MAPK ) and p65 nuclear factor Kappa B (NF‐κB ) in PBMC of AP patients were detected by Western blotting .The co‐localization ,expression and binding between CARD9 and Bcl‐10 in PBMC of control group ,SAP group and MAP group on the Day 1st hospitalization were determined by cell immune‐fluorescence staining and co‐immuno precipitation method .Single factor analysis of variance and Mann‐Whitney test were performed for data comparison between groups .Pearson method was used for correlation analysis .Results The results of Western blotting indicated that the expression of CARD9 and Bcl‐10 in PBMC of SAP group on the Day 1st ,3rd and 5th of hospitalization (1 .12 ± 0 .05 ,1 .03 ± 0 .03 and 1 .01 ± 0 .01 ;1 .74 ± 0 .08 ,1 .72 ± 0 .10 and 1 .69 ± 0 .11) were all significantly higher than those of control group (0 .33 ± 0 .10 and 1 .02 ± 0 .11) and MAP group (0 .71 ± 0 .02 ,0 .55 ± 0 .06 and 0 .25 ± 0 .07 ;1 .15 ± 0 .03 ,1 .09 ± 0 .07 and 1 .01 ± 0 .04) ,and the differences were statistically significant (F= 35 .76 and 18 .20 ,all P< 0 .05) .The expression of p38 MAPK in PBMC of SAP group on the Day lst ,3rd of hospitalization (1 .88 ± 0 .08 、1 .68 ± 0 .11) were significantly higher than those of MAP group on the Day 1st ,3rd ,5th (0 .86 ± 0 .08 ,0 .77 ± 0 .10 ,0 .73 ± 0 .20) and healthy control group (0 .58 ± 0 .24 , F= 7 .24 ,all P < 0 .01) .The expression of p65 NF‐κB in PBMC of SAP group on the Day 1st ,3rd of hospitalization (1 .64 ± 0 .02 ,1 .55 ± 0 .03) were significantly higher than those of MAP group on the Day 3rd ,5th (1 .06 ± 0 .14 ,0 .87 ± 0 .20) and healthy control group (1 .17 ± 0 .13 ,F= 4 .51 ,all P< 0 .05) .The results of immune‐fluorescence staining indicated that CARD9 and Bcl‐10 co‐localized in nucleus .The results of co‐immuno precipitation showed that the binding degree between CARD9 and Bcl‐10 of SAP group was significantly higher than that of control group and MAP group . Pearson correlative analysis suggested that the level of p65 NF‐κB and p38 MAPK in PBMC of AP patients were positive correlated with the expression of CARD9 (r= 0 .692 and 0 .834 ,both P< 0 .01) .Conclusion CARD9 is positive correlated with NF‐κB and MAPK , which indicates CARD9 induced inflammatory cytokines by activating NF‐κB and MAPK signaling pathways in AP .

Roles of UⅡ/UT system played in innate immune inflammatory signal pathway TLR4-IRF3 in LPS-stimulated primary Kupffer cells / 中国免疫学杂志

Objecitve:To investigate effects of urotensin Ⅱ( UⅡ)/UT system on innate immune inflammatory signal pathway TLR4-IRF3 in the lipopolysaccharide (LPS)-stimulated Kupffer cells (KCs).Methods: Rat KCs were isolated and cultured.Pro-in-flammatory cytokines including IL-6,IFN-βand IFN-γwere assayed by ELISA in culture supernatant of KCs.Cell surface TLR4 were tested with flow cytometry technique.Expression of IRF3 were tested with real-time PCR and Western blot.Results: Significant increases were showed in IL-6, IFN-βand IFN-γsecretion, TLR4-expressed positive rates and IRF3 mRNA levels in KCs after stimulated by LPS,but were inhibited via urantide pretreatment.In addition,LPS induced upregulation of nuclear IRF3 protein and downregulation of cytoplasm IRF3 protein in KCs,which were blocked by urantide pretreatment.Conclusion:UⅡ/UT system mediates immune inflammatory response in part through activating TLR 4-IRF3 pathway in LPS-stimulated KCs.

Effects of UⅡ/UT system on the expression of inflammatory signal molecules p38 MAPK and NF-κB in LPS-stimulated Kupffer cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of urotensin Ⅱ/urotensin Ⅱreceptor ( UⅡ/UT) system on the expression of inflammatory signal molecules p 38 mitogen-activated protein kinase ( p38 MAPK) and nuclear factor-κB ( NF-κB ) in lipopolysaccharide ( LPS )-stimulated Kupffer cells ( KCs ) . Methods Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifuga-tion.The isolated cells were randomly divided into six treatment groups including group 1:UⅡ(-) urantide (-)LPS(-), group 2:UⅡ(+)urantide(-)LPS(-), group 3: UⅡ(-)urantide(+)LPS(-), group 4:UⅡ(-)urantide(-)LPS(+), group 5:UⅡ(+) urantide(-) LPS(+) and group 6:UⅡ(-)urantide(+) LPS(+) .Western blot assay was performed to detect p 38 MAPK/p-p38 MAPK protein and NF-κB p65 sub-unit.The DNA-binding activity of NF-κB was tested by electrophoretic mobility shift assay (EMSA).Re-sults There was no significant difference with the expression of p 38 MAPK protein in KCs among the six groups (P>0.05).The expression of p65 protein and p-p38 MAPK and the DNA-binding activity of NF-κB were significantly enhanced in LPS-stimulated KCs from groups 4, 5 and 6 in comparison with those in group 1 (P0.05), but that were decreased in group 6 than those in group 4 (all P<0.01).Conclusion UⅡ/UT system participated in the activation of p38 MAPK and NF-κB signaling pathways in LPS-stimulated primary Kupffer cells .

Significance of transrectal ultrasound combined with carcinoembryonic antigen examination on accurate preoperative staging of rectal carcinoma / 肿瘤研究与临床

Objective To study the consistency of preoperative staging by transrectal ultrasonography (TRUS) combined with serum carcinoembryonic antigen (CEA) and the postoperative pathological TNM staging (PTNM).Methods 118 rectal cancer patients pathologically proven were divided into preoperative TRUS combined with CEA group (59 cases) and along TRUS group (59 cases).The consistency of preoperative stag in 2 groups and postoperative pathological stage was analyzed retrospectively.Results In TRUS combined with CEA group,the accuracy of T stage was 79.7 ％ (47/59),and the accuracy of N stage was 77.8 ％ (42/59),compared with the postoperative pTNM.While in along TRUS group,the accuracy of T stage was 86.4 ％ (51/59),and the accuracy of N stage was 57.7 ％ (30/59).The consistencies of T and N stage in TRUS combined with CEA group and postoperative pTNM were better (κ =0.685,P =0.000; κ =0.544,P =0.000).While the consistency of T stage in along TRUS and postoperative pTNM was better (κ =0.755,P =0.000),but that of N stage was poor (κ =0.154,P =0.229).Conclusion Preoperative evaluation by the TRUS combined with CEA can increase the accuracy of preoperative stage which can provide more reliable basis for decision-making and improve the rate of coincidence of operative procedures in line with forecasts.At the same time,it can provide the basis for the accurate preoperative diagnosis and individualized treatment.

Preparation, characterization and content determination of cubic phase gel containing capsaicin / 中国中药杂志

<p><b>OBJECTIVE</b>To prepare the cubic phase gel containing capsaicin and characterize its properties.</p><p><b>METHOD</b>The cubic phases gel composed of glycerol monoolein, capsaicin and water was made by self-emulsion technology. The characterization of cubic phase gel was carried out by cross-polarizing light microscopy (CPLM) and Small Angle X-Ray Scattering (SAXS). The capsaicin content was determined by HPLC analysis.</p><p><b>RESULT</b>Under CPLM, cubic phase gel showed dark background. SAXS scattering spectra showed the scattering peaks at 0.1096, 0.1334, 0.1557, 0.1883 A(-1) which was compatible with q1:q2:q3:q4 = mean square root of 2: mean square root of 3: mean square root of 4: mean square root of 6. It was well known that the scattering vector ratio was the characteristic of cubic phase and the internal structure was confirmed to be Pn3m (Q224). The linear range for capsaicin determination was 3.25 x 10(-4) - 2.08 x 10(-2) g x L(-1) (R2 = 1). The average recovery was 97.53% with RSD of 2.9% (n=9).</p><p><b>CONCLUSION</b>CPLM and SAXS technology are suitable to characterize the cubic phase gel The determination of the capsaicin content by HPLC is simple and reproducible.</p>

Study On Optimal Extraction Technology for Volatile Oil in Radix Angelicae Pubescentis / 中药新药与临床药理

Objective To study the optimal extraction process of volatile oil in Radix Angelicae Pubescentis.Methods L9(34) orthogonal design was carried out for the optimization of water extraction of volatile oil in Radix Angelicae Pubescentis,and the content of volatile oil was used as referece index.Results The optimal extraction process was as follows:medicinal materials were crashed to go through mesh 80～100,then were soaked in 12-fold volume of water for 10 hours,and were extracted for 11 hours.Conclusion The optimal extraction techndogy is simple and feasible with the extraction rate of volatile oil being 2.66 %.