Low host immune pressure may be associated with the development of hepatocellular carcinoma: a longitudinal analysis of complete genomes of the HBV 1762T, 1764A mutant.

Background: It has been reported that hepatitis B virus (HBV) double mutations (A1762T, G1764A) are an aetiological factor of hepatocellular carcinoma (HCC). However, it is unclear who is prone to develop HCC, among those infected with the mutant. Exploring HBV quasispecies, which are strongly influenced by host immune pressure, may provide more information about the association of viral factors and HCC. Materials and methods: Nine HCC cases and 10 controls were selected from the Long An cohort. Serum samples were collected in 2004 and 2019 from subjects with HBV double mutations and the complete genome of HBV was amplified and sequenced using next-generation sequencing (NGS). Results: The Shannon entropy values increased from 2004 to 2019 in most cases and controls. There was no significant difference in mean intrahost quasispecies genetic distances between cases and controls. The change in the values of mean intrahost quasispecies genetic distances of the controls between 2004 and 2019 was significantly higher than that of the cases (P<0.05). The viral loads did not differ significantly between cases and controls in 2004(p=0.086) but differed at diagnosed in 2019 (p=0.009). Three mutations occurring with increasing frequency from 2004 to 2019 were identified in the HCC cases, including nt446 C→G, nt514 A→C and nt2857T→C. Their frequency differed significantly between the cases and controls (P<0.05). Conclusions: The change in the values of mean intrahost quasispecies genetic distances in HCC was smaller, suggesting that HBV in HCC cases may be subject to low host immune pressure. Increasing viral loads during long-term infection are associated with the development of HCC. The novel mutations may increase the risk for HCC.

Ethanol Extract of Citrus grandis 'Tomentosa' Exerts Anticancer Effects by Targeting Skp2/p27 Pathway in Non-Small Cell Lung Cancer.

SCOPE: This study aims to investigate the anticancer properties of Citrus grandis 'Tomentosa' (CGT) in non-small cell lung cancer (NSCLC). METHODS AND RESULTS: The ethanol extract of CGT (CGTE) is prepared by using anhydrous ethanol and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), revealing that the main chemical components in CGTE are flavonoids and coumarins, such as naringin, rhoifolin, apigenin, bergaptol, and osthole. CGTE at concentrations without inducing cell death significantly inhibits cell proliferation via inducing cell cycle G1 phase arrest by MTT, colony formation, and flow cytometry assays, implying that CGT has anticancer potential. CGTE markedly inhibits the activity of Skp2-SCF E3 ubiquitin ligase, decreases the protein level of Skp2, and promotes the accumulation of p27 by co-immunoprecipitation (co-IP) and in vivo ubiquitination assay; whereas Skp2 overexpression rescues the effects of CGTE in NSCLC cells. In subcutaneous LLC allograft and A549 xenograft mouse models, CGTE, without causing obvious side effects in mice, significantly inhibits lung tumor growth by targeting the Skp2/p27 signaling pathway. CONCLUSION: These findings demonstrate that CGTE efficiently inhibits NSCLC proliferation both in vitro and in vivo by targeting the Skp2/p27 signaling pathway, suggesting that CGTE may serve as a therapeutic candidate for NSCLC treatment.

A novel subgenotype I3 of hepatitis B virus in Guangxi, China: a 15-year follow-up study.

Genotype I of hepatitis B virus (HBV) was proposed recently following sequencing of complete HBV genomes from Vietnam and Laos. However, its long-term molecular evolution is unknown. The objectives of this study were to study the molecular evolution of this genotype from an asymptomatic HBsAg carrier from the Long An cohort over a 15-year period was studied using both NGS and clone-based sequencing. The number of complete genome sequences obtained in 2004, 2007, 2013, and 2019 are 17, 20, 19, and 10, respectively. All strains belong to subgenotype I1, except for six (five from 2007 and one from 2019) and 8 further strains from 2007 which form a cluster branching out from other subgenotype I sequences, supported by a 100% bootstrap value. Based on complete genome sequences, all of the estimated intragroup nucleotide divergence values between these strains and HBV subgenotypes I1-I2 exceed 4%. These strains are recombinants between genotype I1 and subgenotype C but the breakpoints vary. The median intrahost viral evolutionary rate in this carrier was 3.88E-4 substitutions per site per year. The Shannon entropy (Sn) ranged from 0.55 to 0.88 and the genetic diversity, D, ranged from 0.0022 to 0.0041. In conclusion, our data provide evidence of novel subgenotypes. Considering that the 8 strains disappeared after 2007, while one of the 6 strains appears again in 2019, we propose these 6 strains as a new subgenotype, provisionally designated HBV subgenotype I3 and the 8 strains as aberrant genotype.

Clonorchis sinensis infection induces hepatobiliary injury via disturbing sphingolipid metabolism and activating sphingosine 1-phosphate receptor 2.

Clonorchis sinensis (C. sinensis) infection induces severe hepatobiliary injuries, which can cause inflammation, periductal fibrosis, and even cholangiocarcinoma. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) and its receptor S1P receptors (S1PRs) have been implicated in many liver-related diseases. However, the role of S1PRs in C. sinensis-mediated biliary epithelial cells (BECs) proliferation and hepatobiliary injury has not been elucidated. In the present study, we found that C. sinensis infection resulted in alteration of bioactive lipids and sphingolipid metabolic pathways in mice liver. Furthermore, S1PR2 was predominantly activated among these S1PRs in BECs both in vivo and in vitro. Using JTE-013, a specific antagonist of S1PR2, we found that the hepatobiliary pathological injuries, inflammation, bile duct hyperplasia, and periductal fibrosis can be significantly inhibited in C. sinensis-infected mice. In addition, both C. sinensis excretory-secretory products (CsESPs)- and S1P-induced activation of AKT and ERK1/2 were inhibited by JTE-013 in BECs. Therefore, the sphingolipid metabolism pathway and S1PR2 play an important role, and may serve as potential therapeutic targets in hepatobiliary injury caused by C. sinensis-infection.

Epidemiology and determinants of Clonorchis sinensis infection: A community-based study in southeastern China.

Clonorchiasis is caused by the ingestion of raw freshwater fish and causes high burden in East Asia. The epidemiological profiles and determinants of C. sinensis infection, knowledge, practice and attitude related to clonorchiasis were demonstrated in Hengxian county, Guangxi, China. The overall prevalence of C. sinensis was 60.3%, which was higher in male than in female and in elder population compared to children. The percentage knowing local transmission, transmission route and harm of C. sinensis was 20.9%, 31.4% and 32.5%. A percentage of 60.4% reported ingestion of raw freshwater fish, while 70.1% persons would like to accept treatment if infected with C. sinensis. Of the individuals ingesting raw freshwater fish, 82.0% did at home and 81.3% drunk alcohol when ingesting raw freshwater fish. Sixty-two percent showed the persistence on ingestion of raw freshwater fish in future. The ingestion of raw freshwater fish varied by seasons, with a proportion of 57.8% in spring, 48.3% in summer, 60.2% in autumn and 92.2% in winter. In multivariable regression models, age groups, history with C. sinensis treatment, and frequency on ingesting raw freshwater fish were related to C. sinensis infection, while gender, educational level, history with C. sinensis infection, and knowing local transmission were related to the practice of ingesting raw freshwater fish. Gender, history with C. sinensis treatment, frequency on ingesting raw freshwater fish, duration of ingesting raw freshwater fish and drinking alcohol when ingesting raw freshwater fish were associated to the attitude to the ingestion of raw freshwater fish in future. Great efforts are needed to combat clonorchiasis, in which the heterogeneity of population in knowledge, practice, attitude and infection needs to be considered. Men are of crucial importance in term of the morbidity control through chemotherapy. Meanwhile, massive education is expected to implement, which needs to promote the change of ingesting raw freshwater fish for sustainable control of clonorchiasis.

Severe hepatobiliary morbidity is associated with Clonorchis sinensis infection: The evidence from a cross-sectional community study.

Clonorchis sinensis infection is highly prevalent in Asia. Diverse hepatobiliary morbidity has been documented for C. sinensis infection. This study aimed to assess the association between C. sinensis infection and hepatobiliary morbidity, taking into consideration of the control, confounders and infection intensity. A cross-sectional community survey was implemented in Hengxian county, southeastern China. Helminth infections were detected by fecal examination. Physical examination and abdominal ultrasonography were then conducted. After excluding confounding effects from gender, age and alcohol drinking, quantitative association between C. sinensis infection and hepatobiliary morbidity was assessed, and the effect from infection intensity was also evaluated, through adjusted odds ratio (aOR) and 95% confidence intervals (95% CI). 696 villagers older than 10 years were enrolled. The prevalence and infection intensity of C. sinensis were higher in male, elder people and the individuals consuming alcohol. Light C. sinensis infection was associated with the increase of diarrhoea (aOR: 2.2, 95% CI: 1.1-4.5). C. sinensis infection was associated with the increase of fatty liver (aOR: 2.7, 95% CI: 1.4-5.2), and the effect was similar in different infection intensities. Moderate C. sinensis infection was associated with the increase of gallbladder stone (aOR: 3.0, 95% CI: 1.1-8.6), while moderate and heavy infections with the increase of intrahepatic bile duct dilatation (aOR: 2.2, 95% CI: 1.0-4.9 and aOR: 4.3, 95% CI: 1.9-9.9, respectively). C. sinensis infection had an effect on the development of periductal fibrosis (aOR: 3.2, 95% CI: 2.1-4.9), which showed increasing trend by infection intensity. The length and width of gallbladder in those with C. sinensis infection were enlarged, especially in those over 30 years old. C. sinensis infection is significantly associated with hepatobiliary morbidity. The occurrence of some morbidity was strongly related to the infection intensity. Awareness on harm of clonorchiasis should be raised both for policy-makers and villagers to adopt effective interventions.

High prevalence of Clonorchis sinensis infection in Guangxi, Southern China.

BACKGROUND: Soil-transmitted helminths (STHs), such as hookworm, roundworm and whipworm, and food-borne trematodiases, including Clonorchis sinensis, remain a public health problem worldwide, especially in tropical and subtropical regions. OBJECTIVE: We aimed to determine the current prevalence of these parasites in Guangxi, China, which is located in a subtropical region. METHODS: A cross-sectional study and a 4-year longitudinal surveillance study were carried out. Stool samples were collected and examined microscopically for parasite eggs using the modified Kato-Katz thick smear method. RESULTS: The study subjects selected using stratified random cluster sampling for the cross-sectional study and longitudinal surveillance study numbered 15,683 and 24,429, respectively. In the cross-sectional study, hookworm, roundworm, whipworm, pinworm, C. sinensis, and tapeworm were found. The total prevalence of soil-transmitted helminths (STHs) was 6.4% (95% CI, 6.0-6.8). The prevalences of C. sinensis, hookworm, roundworm, whipworm, and pinworm were 10.6%, 4.2%, 0.3%, 0.3%, and 1.8%, respectively. The prevalence of C. sinensis in males (14.0%, 95% CI, 13.3-14.8) was significantly higher than in females (7.2%, 95% CI, 6.7-7.8) (P = 0.0001). The prevalence also was significantly higher in the medical worker group (20.8%, 95% CI, 12.9-28.7) than in all other occupational groups (10.5%, 95% CI, 10.0-11.0) (P = 0.0001). The prevalence of hookworm in females (5.3%, 95% CI, 4.8-5.8) was significantly higher than in males (3.0%, 95% CI, 2.6-3.3) (P = 0.0001). In the longitudinal surveillance study, the prevalence of C. sinensis and STHs in 2016, 2017, 2018, and 2019 were 12.0%, 6.0%, 11.0%, and 10.0% and 2.6%, 2.8%, 1.5%, and 1.5%, respectively. CONCLUSIONS: Adult male and occupation of and medical workers are risk factors for infection with C. sinensis and hookworm. The prevalence rate of C. sinensis remains high while those of the other STHs are decreasing, suggesting that enhanced health education should be focused on C. sinensis in Guangxi.

Familial assimilation in transmission of raw-freshwater fish-eating practice leading to clonorchiasis.

Clonorchiasis is caused by raw-freshwater fish-eating practice and causes high burden in Asia. Transmission mechanism of this behavior hasn't been illuminated, which hinders the adoption of sustainable control activities. A cross-sectional survey was implemented in students from four endemic provinces in China. Data with 23,222 students aged 9-18 and their parents were eligible. Familial clustering of raw-eating practice, impact of parents' practice on children, interaction of spouses' practice was analyzed. Raw-eating practice met ß-binomial distribution (χ2 = 0.8, p>0.05). Clustering coefficient increased by students' age (R2 = 0.82, p<0.001) and was higher in those families with boys compared to girls (t = 4.1, p<0.01). The proportion of students with raw-eating practice increased yearly by 8.9% in girls and 10.5% in boys. Compared to those without parents' raw-eating practice, adjusted odds ratio of students' raw-eating practice was 10.5 (95% confidential intervals (95% CI): 9.4-11.7) in those with fathers' practice, 33.6 (95% CI: 26.3-42.9) in those with mothers' practice and 47.1 (95% CI: 42.0-52.8) in those with both parents' practice. There existed interaction between spouses' practice (χ2 = 6713.1, p<0.001) and the impact from husband on his wife was higher than that from wife on her husband. Familial assimilation characterizes the transmission of raw-freshwater fish-eating practice, consisted of vertical intergenerational assimilation from parents to their children and horizontal martial assimilation between spouses. A sustainable strategy against clonorchiasis should interrupt the transmission of raw-freshwater fish-eating practice. Additionally, further studies are expected to explore more information, e.g. the frequency in raw-eating practice and type of raw freshwater fish, infection status of C. sinensis in participants, as well as direct collection of parents' eating information from themselves.

Unmasking the heterogeneity of carbohydrates in heartwood, sapwood, and bark of Eucalyptus.

In this study, the cellulose and hemicelluloses in heartwood, sapwood, and bark of E. urophylla × E. grandis were comprehensively investigated. The ultrastructural topochemistry of carbohydrates in cell walls was examined in situ by confocal Raman microscopy. Cellulose and alkali-extractable hemicelluloses samples were isolated from different tissues and comparatively characterized by compositional carbohydrate analyses, determination of molecular weights, FT-IR spectroscopy, and XRD and NMR techniques. It was found that among all of the samples, heartwood cellulose had the highest molecular weight as well as the lowest degree of crystallinity. Meanwhile the hemicelluloses in heartwood had higher xylose content, lower degree of branching, slightly lower molecular weights but narrower polydispersity than those in sapwood. The eucalyptus hemicelluloses mainly consisted of (1→4)-ß-D-xylan backbone with glucuronic acid side chains. Furthermore, the hemicelluloses isolated from sapwood had a higher degree of substitution with terminal galactose than those isolated from heartwood and bark.

Effectiveness of health education in improving knowledge, practice and belief related to clonorchiasis in children.

Clonorchiasis is caused by the ingestion of raw freshwater fish containing Clonorchis sinensis larvae. Health education is crucial in sustainable control of clonorchiasis but extremely challenging when dealing with adults. To investigate if education in children would be a beneficial long-term approach, a cluster-controlled trial was implemented in two primary schools between 2017 and 2019 in Binyang county, Guangxi, China. A baseline survey was conducted in both schools, and then comprehensive education activities were implemented in the interventional one. Six and 12 months later, all pupils were subjected to a survey to assess the potential change of knowledge, practice and belief related to clonorchiasis. Finally, 247 pupils in interventional school and 151 ones in control were eligible for analysis. In the interventional school, the percentage of pupils with knowledge on transmission route, early symptoms, complications and carcinogenicity increased from 15.0% to 78.5% (p < 0.001), from 6.5% to 36.8% (p < 0.001), from 4.0% to 93.9% (p < 0.001) and from 4.5% to 49.4% (p < 0.001) after 12 months. In this school, 16 pupils (6.5%) had eaten raw freshwater fish during the year immediately before the baseline survey, a number that decreased to 3 (1.2%) (p = 0.002) 12 months later. The percentage of students had the belief on the ability not to eat raw freshwater fish in the future was 70.9% at the baseline and 97.2% 12 months later (p < 0.001). Although knowledge related to clonorchiasis also increased in the control school, the absolute percentage of changes was lower; in particular, practice and belief had not changed.

Rapid screening of Clonorchis sinensis infection: Performance of a method based on raw-freshwater fish-eating practice.

Clonorchis sinensis infection is caused by ingestion of raw freshwater fish containing the infective larvae of Clonorchis sinensis. It is highly endemic in East Asia, especially in China. Selective chemotherapy of people who report habitual eating of raw freshwater fish is a control measure. As the performance of this screening technique has not yet been fully evaluated in China, a cross-sectional study was conducted, covering 17 counties in four major clonorchiasis-endemic provinces. About 1 000 participants were enrolled from each county. Fecal samples were collected and examined for helminth eggs and each person enrolled was asked about their practice with respect to eating raw freshwater fish. In total, 16 230 participants from 16 counties were finally included. The overall prevalence of C. sinensis infection was 10.8%, ranging from 0 to 53.7% in the 16 counties, while the percentage of inhabitants eating raw freshwater fish was 26.5%, ranging from 0 to 79.1%. The overall sensitivity and specificity of screening for C. sinensis infection in this approach was 82.3% and 80.3%, respectively, yielding a Youden's index of 0.6. The overall positive and negative likelihood ratios were 4.2 and 0.2, respectively, while the overall positive and negative predictive values were 33.5% and 97.4%, respectively. Furthermore, the sensitivity was higher with regard to high-intensity infections compared to light infections.

Structurally diverse diterpenoids from Trigonostemon howii.

Phytochemical investigation on the stems and leaves of Trigonostemon howii resulted in the isolation of a new abietane diterpenoid, trigohowimine A (1), along with seven known structurally diverse diterpenoids (2-8). The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparison with data reported in the literature. New compound 1 was evaluated for its cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480. Compound 1 showed significant inhibitory effects against various human cancer cell lines with IC50 values ranging from 0.82 to 8.53 µM.

Furanocoumarins with potential antiproliferative activities from Clausena lenis.

A phytochemical investigation on the stems and leaves of Clausena lenis led to the isolation of a new furanocoumarin, clauselenisin A (1), together with five known analogues (2-6). The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons with data reported in the literature. All known compounds (2-6) were isolated from C. lenis for the first time. All isolated compounds were evaluated for their their antiproliferative activities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Compounds 1-6 showed significant antiproliferative effects with IC50 values ranging from 0.36 to 16.48 µM.

Carbazole alkaloids from Clausena emarginata with their potential antiproliferative activities.

A phytochemical investigation on the stems and leaves of Clausena emarginata led to the isolation of a previously undescribed carbazole alkaloid, clausemargine A (1), together with 11 known analogues (2-12). The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons with data reported in the literature. All known compounds (2-12) were isolated from C. emarginata for the first time. All isolated compounds were evaluated for their their antiproliferative activities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Compounds 1-12 showed significant antiproliferative effects with IC50 values ranging from 0.28 to 15.18 µM.

Novel γ-lactone derivatives from Trigonostemon heterophyllus with their potential antiproliferative activities.

Two novel γ-lactone derivatives, trigoheterophines A (1) and B (2), together with four known furan derivatives (3-6), were isolated from the stems and leaves of Trigonostemon heterophyllus. The structures of 1 and 2 were elucidated by extensive spectroscopic methods and the known compounds were identified by comparing with the data reported in literature. Among them, trigoheterophines A (1) and B (2) represent an unusual type of γ-lactone derivatives, possessing 21 carbon atoms on the carbon skeleton, and known compouds (3-6) are rare furan derivatives in the plant kingdom with diverse long-chain hydrocarbyl groups as substituents at C-4. All isolated compounds were evaluated for their antiproliferative activities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Compounds 1-6 showed significant antiproliferative effects against various human cancer cell lines with IC50 values ranging from 0.28 to 12.06 µM. These findings suggest that the discoveries of these novel γ-lactone derivatives and furan derivatives with significant antiproliferative activities isolated from T. heterophyllus could be of great importance to the development of new anticancer agents.

Performance evaluation of existing immunoassays for Clonorchis sinensis infection in China.

BACKGROUND: Clonorchiasis ranks among the most important food-borne parasitic diseases in China. However, due to low compliance to traditional fecal examination techniques in the general population and medical personnel, immunodiagnosis is expected. This study evaluated, in parallel, the performance of four immunodiagnostic kits detecting clonorchiasis in China. RESULTS: A bank with 475 sera was established in this study. Except for the low performance of the kit detecting IgM, the other three kits detecting IgG showed sensitivities ranging from 81.51% (194/238) to 99.16% (236/238). Higher sensitivity was presented in heavy infection intensity [89.47% (68/76) to 100% (76/76)]. Among the four kits, the overall specificity varied from 73.42% (174/237) to 87.34% (207/237). It was observed that the specificity was lower in the sera of the participants living in clonorchiasis-endemic areas but without any parasite infection [67.5% (81/120) to 90% (108/120)], as compared to those from the non-endemic area [94% (47/50) to 98% (49/50)]. The cross-reaction rate varied from 14.93% (10/67) to 31.34% (21/67). Youden's index was -0.022, 0.689, 0.726, and 0.802 for kits T1, T2, T3 and T4, respectively. Repeatability was high in all four kits. CONCLUSIONS: Three immunodiagnosis kits targeting IgG antibody had high performance on detecting chronic Clonorchis sinensis infection, but that detecting IgM antibody had not. The kits detecting IgG antibody also showed high sensitivity in heavy infection intensity. Research on immunological diagnosis of clonorchiasis is expected to be strengthened to improve the sensitivity in light infection and specificity.

A new abietane diterpenoid from the roots of Tripterygium regelii.

A new abietane diterpenoid, tripterregeline A (1), together with six known diterpenoids (2-7), were isolated from the roots of Tripterygium regelii. The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparison with data reported in the literature. All isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Compounds 1-7 showed significant inhibitory effects against various human cancer cell lines with IC50 values ranging from 0.58 to 21.06 µM.

Cytotoxic dihydrobenzofuran neolignans from Mappianthus iodoies.

Three new dihydrobenzofuran neolignans, mappiodoinins A-C (1-3), together with nine known analogues (4 -12) were isolated from the stems and leaves of Mappianthus iodoies. Their structures with the absolute configurations were elucidated by extensive spectroscopic methods. This is the first time to find dihydrobenzofuran neolignans from the genus Mappianthus. All isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW-480 in vitro. Neolignans 1-12 showed significant cytotoxic effects against various human cancer cell lines with IC50 values ranging from 0.16 to 18.62 µM.

TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis.

BACKGROUND: Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal. METHODS: TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry. RESULTS: Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t (5) = 2.595, P = 0.049), 60 (t (5) = 7.838, P = 0.001) and 90 dpi (t (5) = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t (5) = 5.139, P = 0.004), 60 (t (5) = 6.138, P = 0.002) and 90 dpi (t (5) = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t (5) = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t (5) = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t (5) = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi. CONCLUSIONS: These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.

A new indole alkaloid with anti-inflammatory activity from Nauclea officinalis.

A new indole alkaloid, 17-O-methyl-19-(Z)-naucline (1), together with seven known alkaloids (2-8), were isolated from the stems and leaves of Nauclea officinalis. The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons their data with those reported in the literature. 17-O-methyl-19-(Z)-naucline (1) showed significant inhibitory activity on nitric oxide production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro with an IC50 value of 3.6 µM.