Characterizing neuroinflammation and identifying prenatal diagnostic markers for neural tube defects through integrated multi-omics analysis.

BACKGROUND: Neural Tube Defects (NTDs) are congenital malformations of the central nervous system resulting from the incomplete closure of the neural tube during early embryonic development. Neuroinflammation refers to the inflammatory response in the nervous system, typically resulting from damage to neural tissue. Immune-related processes have been identified in NTDs, however, the detailed relationship and underlying mechanisms between neuroinflammation and NTDs remain largely unclear. In this study, we utilized integrated multi-omics analysis to explore the role of neuroinflammation in NTDs and identify potential prenatal diagnostic markers using a murine model. METHODS: Nine public datasets from Gene Expression Omnibus (GEO) and ArrayExpress were mined using integrated multi-omics analysis to characterize the molecular landscape associated with neuroinflammation in NTDs. Special attention was given to the involvement of macrophages in neuroinflammation within amniotic fluid, as well as the dynamics of macrophage polarization and their interactions with neural cells at single-cell resolution. We also used qPCR assay to validate the key TFs and candidate prenatal diagnostic genes identified through the integrated analysis in a retinoic acid-induced NTDs mouse model. RESULTS: Our analysis indicated that neuroinflammation is a critical pathological feature of NTDs, regulated both transcriptionally and epigenetically within central nervous system tissues. Key alterations in gene expression and pathways highlighted the crucial role of STATs molecules in the JAK-STAT signaling pathway in regulating NTDs-associated neuroinflammation. Furthermore, single-cell resolution analysis revealed significant polarization of macrophages and their interaction with neural cells in amniotic fluid, underscoring their central role in mediating neuroinflammation associated with NTDs. Finally, we identified a set of six potential prenatal diagnostic genes, including FABP7, CRMP1, SCG3, SLC16A10, RNASE6 and RNASE1, which were subsequently validated in a murine NTDs model, indicating their promise as prospective markers for prenatal diagnosis of NTDs. CONCLUSIONS: Our study emphasizes the pivotal role of neuroinflammation in the progression of NTDs and underlines the potential of specific inflammatory and neural markers as novel prenatal diagnostic tools. These findings provide important clues for further understanding the underlying mechanisms between neuroinflammation and NTDs, and offer valuable insights for the future development of prenatal diagnostics.

Symptom effects and central mechanism of acupuncture in patients with functional gastrointestinal disorders: a systematic review based on fMRI studies.

BACKGROUND: Functional gastrointestinal disorders (FGIDs) are closely related to disorders of brain-gut interaction. FGIDs are the dominant disease of acupuncture treatment, which can improve the symptoms and emotional state. AIM: To evaluate the results and quality of the available clinical evidence and to summarize the central mechanism and effect of acupuncture on FGIDs. METHODS: PubMed, EMBASE, Web of science, Cochrane Library, China National Knowledge Infrastructure (CNKI) were searched by computer to collect the randomized controlled trials (RCTs), which contained central mechanisms via fMRI research of acupuncture in the treatment of FGIDs patients. The search time limit was from the establishment of the database to June 22, 2022. Two researchers independently screened the literature, extracted data, and evaluated the quality. RESULTS: Ten RCTs involving fMRI data were included in this study, including 4 Functional dyspepsia (FD) studies, 3 irritable bowel syndrome (IBS) studies, and 3 functional constipation (FC) studies. The score of improvements in both gastrointestinal symptoms and psychological symptoms showed that acupuncture could significantly improve the clinical symptoms of FGIDs patients, including abdominal pain, abdominal distension, frequency of defecation, and stool characteristics, and could relieve anxiety and depression symptoms of patients. Acupuncture could regulate brain functional connections and functional activity in FGIDs patients, mainly including insula, anterior cingulate cortex, prefrontal cortex, thalamus, hippocampus, amygdala and other brain regions. CONCLUSION: Acupuncture can improve gastrointestinal symptoms and psychological status in FGIDs patients, and regulate functional connectivity and activity of brain regions such as insula, ACC, PFC, thalamus, HIPP, amygdala, etc. These changes in brain activity may related to visceral sensation, pain regulation, emotion, but further studies of high quality are still necessary.

Hypomethylation of GDNF family receptor alpha 1 promotes epithelial-mesenchymal transition and predicts metastasis of colorectal cancer.

Tumor metastasis is the major cause of poor prognosis and mortality in colorectal cancer (CRC). However, early diagnosis of highly metastatic CRC is currently difficult. In the present study, we screened for a novel biomarker, GDNF family receptor alpha 1 (GFRA1) based on the expression and methylation data in CRC patients from The Cancer Genome Altlas (TCGA), followed by further analysis of the correlation between the GFRA1 expression, methylation, and prognosis of patients. Our results show DNA hypomethylation-mediated upregulation of GFRA1 in invasive CRC, and it was found to be correlated with poor prognosis of CRC patients. Furthermore, GFRA1 methylation-modified sequences were found to have potential as methylation diagnostic markers of highly metastatic CRC. The targeted demethylation of GFRA1 by dCas9-TET1CD and gRNA promoted CRC metastasis in vivo and in vitro. Mechanistically, demethylation of GFRA1 induces epithelial-mesenchymal transition (EMT) by promoting AKT phosphorylation and increasing c-Jun expression in CRC cells. Collectively, our findings indicate that GFRA1 hypomethylation can promote CRC invasion via inducing EMT, and thus, GFRA1 methylation can be used as a biomarker for the early diagnosis of highly metastasis CRC.

Temporal DNA methylation pattern and targeted therapy in colitis-associated cancer.

DNA methylation plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). However, the global and temporal DNA methylation pattern during initiation and progression of colitis-associated cancer (CAC) are still unknown, including the potential therapeutic strategy of targeting methylation for CAC. In the present study, the global DNA methylation pattern was determined at different time points during CAC using DNA methylation sequencing, followed by the Starburst plot integrating alterations and potential functional prediction analysis. After demonstrating the regulatory role of DNA methyltransferases (DNMTs) on the expression of hub-genes in CRC cells, DNMT inhibitors were administered to treat CAC mice. Our results indicated that 811 genes were hypermethylated at different time points during initiation and progression of CAC. Genes that were downregulated and hypermethylated during CAC, including hub-genes BAD and inositol polyphosphate phosphatase-like 1 (INPPL1), were involved in MAPK signaling pathways, kit receptor signaling pathways, apoptosis and EGF/EGFR signaling pathways. Upregulated DNMTs (DNMT1, DNMT3A and DNMT3B) mediated downregulation and hypermethylation of BAD and INPPL1 in CAC and CRC cells. Low doses of DNMT inhibitors (decitabine (DAC) and azacitidine (AZA)) exerted efficient antitumor effects in CAC, accompanied with upregulation of BAD and INPPL1 expression, and apoptosis induction. In summary, the present study demonstrates the temporal DNA methylation pattern during CAC and provides a novel therapeutic strategy for treating this disease.

SARI suppresses colitis-associated cancer development by maintaining MCP-1-mediated tumour-associated macrophage recruitment.

SARI (suppressor of AP-1, regulated by IFN) impaired tumour growth by promoting apoptosis and inhibiting cell proliferation and tumour angiogenesis in various cancers. However, the role of SARI in regulating tumour-associated inflammation microenvironment is still elusive. In our study, the colitis-dependent and -independent primary model were established in SARI deficiency mice and immuno-reconstructive mice to investigate the functional role of SARI in regulating tumour-associated inflammation microenvironment and primary colon cancer formation. The results have shown that SARI deficiency promotes colitis-associated cancer (CAC) development only in the presence of colon inflammation. SARI inhibited tumour-associated macrophages (TAM) infiltration in colon tissues, and SARI deficiency in bone marrow cells has no observed role in the promotion of intestinal tumorigenesis. Mechanism investigations indicated that SARI down-regulates p-STAT1 and STAT1 expression in colon cancer cells, following inhibition of MCP-1/CCR2 axis activation during CAC development. Inverse correlations between SARI expression and macrophage infiltration, MCP-1 expression and p-STAT1 expression were also demonstrated in colon malignant tissues. Collectively, our results prove the inhibition role of SARI in colon cancer formation through regulating TAM infiltration.

Modulating the Tumor Microenvironment via Oncolytic Viruses and CSF-1R Inhibition Synergistically Enhances Anti-PD-1 Immunotherapy.

Immunotherapy based on the immune checkpoint blockade has emerged as the most promising approach for cancer therapy. However, the proportion of colorectal cancer patients who benefit from immunotherapy is small due to the immunosuppressive tumor microenvironment. Hence, combination immunotherapy is an ideal strategy to overcome this limitation. In this study, we developed a novel combination of CSF-1R (colony-stimulating factor 1 receptor) inhibitor (PLX3397), oncolytic viruses, and anti-PD-1 antibody. Our results demonstrated that the triple treatment synergistically conferred significant tumor control and prolonged the survival of mouse models of colon cancer. Approximately 43% and 82% of mice bearing the CT26 and MC38 tumor, respectively, survived long term following the triple treatment. This combination therapy reprogrammed the immunosuppressive tumor microenvironment toward a CD8+ T cell-biased anti-tumor immunity by increasing T cell infiltration in the tumor and augmenting anti-tumor CD8+ T cell function. Our results provide a robust strategy for clinical combination therapy.

Temporal expression and functional analysis of long non-coding RNAs in colorectal cancer initiation.

Long non-coding RNAs (lncRNAs) have potential applications in clinical diagnosis and targeted cancer therapies. However, the expression profile of lncRNAs in colorectal cancer (CRC) initiation is still unclear. In this study, the expression profiles of lncRNAs and mRNAs were determined by microarray at specific tumour stages in an AOM/DSS-induced primary colon cancer model. The temporal expression of lncRNAs was analysed by K-means clustering. Additionally, weighted correlation network analysis (WGCNA) and gene ontology analysis were performed to construct co-expression networks and establish functions of the identified lncRNAs and mRNAs. Our results suggested that 4307 lncRNAs and 5798 mRNAs are deregulated during CRC initiation. These differential expression genes (DEGs) exhibited a clear correlation with the differential stage of tumour initiation. WGCNA results suggested that a series of hub lncRNAs are involved in regulating cell stemness, colon inflammation, oxidative stress response and cell death at each stage. Among them, lncRNA H19 was up-regulated in colon tumours and correlated with poor patient prognosis. Collectively, we have been the first to demonstrate the temporal expression and function of lncRNAs in CRC initiation. These results provide novel diagnosis and therapy targets for CRC.

Expression of SUMO1P3 Compared with SUMO1 is an Independent Predictor of Patient Outcome in Lung Adenocarcinoma.

BACKGROUND The small ubiquitin-like modifier 1 (SUMO1) and small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3) are long noncoding RNAs (lncRNAs). The prognostic significance of SUMO1 and SUMO1P3 expression in non-small cell lung cancer (NSCLC) remains unclear. This study aimed to use clinical, genetic, and survival data from the Cancer Genome Atlas (TCGA), to analyze the prognostic significance of SUMO1 and SUMO1P3 expression in the two main subtypes of NSCLC, lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). MATERIAL AND METHODS Data were acquired from TCGA and in silico survival analysis was performed. SUMO1 and SUMO1P3 expression were compared between patients with LUAD and LUSC. Patient outcome was assessed as complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). Recurrence-free survival (RFS) was defined as the survival time from primary surgery to the time of locoregional or distant recurrence of lung cancer. RESULTS SUMO1P3 was significantly increased in LUSC and LUAD tissues compared with adjacent normal lung tissue and was significantly co-expressed with SUMO1. SUMO1P3 expression was significantly increased in patients with LUAD but not LUSC with reduced RFS after primary or follow-up treatment. Although patients with LUAD who had high SUMO1 or SUMO1P3 expression had reduced RFS compared with low expression groups, univariate and multivariate analysis showed that only SUMO1P3 expression was independently associated reduced RFS (HR, 1.418; 95% CI, 1.041-1.930; p=0.027). CONCLUSIONS SUMO1P3 expression was an independent indicator of reduced RFS in patients with LUAD.

Follistatin-like protein 5 inhibits hepatocellular carcinoma progression by inducing caspase-dependent apoptosis and regulating Bcl-2 family proteins.

Hepatocellular carcinoma (HCC) is one of the most common and deadly malignant tumors in the world, especially in China. Follistatin-like protein 5 (FSTL5) is a member of the FSTL family, which is involved in cell proliferation, migration, differentiation, and embryo development. We aimed to investigate the function and underlying mechanism of FSTL5 in HCC. FSTL5 expression was determined by immunohistochemistry staining in a liver cancer tissue microarray (TMA) and the correlation between FSTL5 and the prognosis of HCC patients was analysed. Further proliferation assay, colony formation assay, flow cytometry, and xenograft tumor model were performed to investigate the bioeffects of FSTL5 in HCC in vitro and in vivo. We found that FSTL5 expression was downregulated in HCC tissues and positively correlated with the prognosis of patients with HCC at tumor node metastasis stage I/II. Overexpression of FSTL5 efficiently impaired HCC growth both in vivo and in vitro with an exogenous manner. Mechanistic investigation demonstrated that FSTL5 promoted HCC cell apoptosis in a caspase-dependent manner and regulated Bcl-2 family proteins. These results indicate that FSTL5 may be a potential novel target for HCC treatment, and a biomarker for tumor prognosis.

IL-35 recombinant protein reverses inflammatory bowel disease and psoriasis through regulation of inflammatory cytokines and immune cells.

Interleukin-35 (IL-35), a member of the IL-12 family, functions as a new anti-inflammatory factor involved in arthritis, psoriasis, inflammatory bowel disease (IBD) and other immune diseases. Although IL-35 can significantly prevent the development of inflammation in many diseases, there have been no early studies accounting for the role of IL-35 recombinant protein in IBD and psoriasis. In this study, we assessed the therapeutic potential of IL-35 recombinant protein in three well-known mouse models: the dextransulfate sodium (DSS)-induced colitis mouse model, the keratin14 (K14)-vascular endothelial growth factor A (VEGF-A)-transgenic (Tg) psoriasis mouse model and the imiquimod (IMQ)-induced psoriasis mouse model. Our results indicated that IL-35 recombinant protein can slow down the pathologic process in DSS-induced acute colitis mouse model by decreasing the infiltrations of macrophages, CD4+ T and CD8+ T cells and by promoting the infiltration of Treg cells. Further analysis demonstrated that IL-35 recombinant protein may regulate inflammation through promoting the secretion of IL-10 and inhibiting the expression of pro-inflammatory cytokines such as IL-6, TNF-α and IL-17 in acute colitis model. In addition, lower dose of IL-35 recombinant protein could achieve long-term treatment effects as TNF-α monoclonal antibody did in the psoriasis mouse. In summary, the remarkable therapeutic effects of IL-35 recombinant protein in acute colitis and psoriasis mouse models indicated that IL-35 recombinant protein had a variety of anti-inflammatory effects and was expected to become an effective candidate drug for the treatment of inflammatory diseases.

Human Adipose-Derived Mesenchymal Stem Cell-Secreted CXCL1 and CXCL8 Facilitate Breast Tumor Growth By Promoting Angiogenesis.

Autologous adipose tissue or adipose tissue with additive adipose-derived mesenchymal stem cells (ADSCs) is used in the breast reconstruction of breast cancer patients who undergo mastectomy. ADSCs play an important role in the angiogenesis and adipogenesis, which make it much better than other materials. However, ADSCs may promote residual tumor cells to proliferate or metastasize, and the mechanism is still not fully understood. In this study, we demonstrated that human ADSCs (hADSCs) could facilitate tumor cells growth after co-injection with MCF7 and ZR-75-30 breast cancer cells (BCCs) by promoting angiogenesis, but hADSCs showed limited effect on the growth of MDA-MB-231 BCCs. Intriguingly, compared with ZR-75-30 tumor cells, MCF7 tumor cells were more potentially promoted by hADSCs in the aspects of angiogenesis and proliferation. Consistent with this, cytokine and angiogenesis array analyses showed that after co-injection with hADSCs, the CXCL1 and CXCL8 concentration were significantly increased in MCF7 tumor, but only moderately increased in ZR-75-30 tumor and did not increase in MDA-MB-231 tumor. Furthermore, we found that CXCL1/8 were mainly derived from hADSCs and could increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs) by signaling via their receptors CXCR1 and CXCR2. A CXCR1/2-specific antagonist (SCH527123) attenuated the angiogenesis and tumor growth in vivo. Our findings suggest that CXCL1/8 secreted by hADSCs could promote breast cancer angiogenesis and therefore provide better understanding of safety concerns regarding the clinical application of hADSCs and suggestion in further novel therapeutic options. Stem Cells 2017;35:2060-2070.

RAMP3 is a prognostic indicator of liver cancer and might reduce the adverse effect of TP53 mutation on survival.

AIM: To assess the prognostic value of RAMP3 expression in terms of overall survival (OS) and recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients. MATERIALS & METHODS: Immunochemistry staining was performed to detect RAMP3 expression. Data in the Cancer Genome Atlas-Liver Hepatocellular Cancer were used for secondary analysis. RESULTS: RAMP3 expression was significantly downregulated in HCC tissues than in normal liver tissues. Increased RAMP3 expression was an independent prognostic factor of favorable OS (hazard ratio [HR]: 0.772, 95% CI: 0.689-0.864; p < 0.001) and RFS (HR = 0.719, 95% CI: 0.633-0.817; p < 0.001). High RAMP3 expression was associated with significantly better RFS in both TP53 mutant and wildtype groups. CONCLUSION: High RAMP3 RNA expression is an independent prognostic factor of favorable OS and RFS in patients with HCC.

Comparative RNA-Seq transcriptome analysis on silica induced pulmonary inflammation and fibrosis in mice silicosis model.

Silicosis is a long-established public health issue in developing countries due to increasingly serious air pollution and poorly implemented occupational safety regulation. Inhalation of silica triggers cytotoxicity, oxidative stress, pulmonary inflammation and eventually silicosis. Current understanding in the pathogenesis and mechanism of silicosis is limited, and no effective cure is clinically available once silicosis is developed. A number of studies were conducted to investigate silica-induced alternate gene expressions in pulmonary cells. However, transcriptome analysis in a silicosis animal model is needed. This study was performed to evaluate the transcriptional alternations in silicotic mice using comparative RNA-Seq. A silicosis mice model was established by intratracheal instillation of silica suspensions, and validated by histological examinations. High-throughput sequencing and differential gene expression analysis revealed 749 upregulated genes and 70 downregulated genes in the silicosis model. Genes related to immune cell interactions, immune cell responses and inflammation were significantly enriched. Cytokine-cytokine receptor interaction and downstream JAK-STAT signaling pathways were the most significantly enriched KEGG pathways. Reverse transcription-polymerase chain reaction analysis and immunohistochemistry were performed to validate further the differential expression patterns of representative genes. The reported results in this study provide the basis for elucidating the molecular mechanisms for silica-induced pulmonary inflammation and fibrosis, and support the prevention and treatment of silicosis.

[Multi-dimensional Clinical Profile for Functional Gastrointestinal Disorders and Diagnostic and Therapeutic Approaches of Chinese Medicine].

Functional gastrointestinal disorders (FGIDs) are manifested as digestive symptoms, but relative symptoms cannot be confirmed by traditional inspection methods. In 2015 Rome Foundation put forward the conception of multi-dimensional clinical profile (MDCP) for FGIDs, which emphasized multi-dimensional assessment of disease state and aimed to develop individualized treatment program. Chinese medicine also has multi-dimensional thoughts in diagnosis and treatment and has much in com- mon with MDCP in refining diagnosis, attaching importance to psychological factors and spirits, seeking biomarkers, and so on. The correlation between multi-dimensional diagnostic and therapeutic thoughts in Chinese medicine and MDCP was explored by combining functional dyspepsia as focal point.

[Effect of Shen warming Pi strengthening method on the expression of serum T cell subsets in IBS-D rats].

OBJECTIVE: To observe the effect of Shen warming Pi strengthening method on expressions of serum T cell subsets (C045+%, C03+%, and C04 +/COB+) in diarrhea-predominant irritable bowel syndrome (IBS-0) rats. Methods An IBS-0 rat model was established referring to AL-Chaer's modeling method combined with tail clamp and intragastric administration of sanna leaf. After modeling 30 SO rats were randomly divided into 6 groups according to random digit table, i.e., the model group, the high, middle, low dose Wenshen Jianpi Recipe (WJR) groups, and the Sishen Pill control group, 6 in each group. A normal control group consisting of 6 SO rats were also set up. Rats in high, middle, low dose WJ R groups were administered by gastrogavage with boil-free WJ R at the daily dose of 3. 100, 1. 550, 0. 775 g/kg, respectively. Rats in the Sis hen Pill control group were administered by gastrogavage with boil-free Sis hen Pill at the daily dose of 0. 736 g/kg. Equal volume of normal saline was given by gastrogavage to rats in the model group and the normal control group. All medication lasted for 2 successive weeks. Rats' general state, expressions of T cell subsets (CD45+%, CD3+%, and CD4+ /CDB+) changes were observed. RESULTS: Compared with the normal control group, expressions of CD45+% and CD3+% increased, but CD4+ /CDB+ decreased with statistical difference (P < 0. 05). Compared with the model group, expressions of CD45+% and CD3+% decreased, but CD4+ ICDB+ increased with statistical difference in high, middle, low dose WJR groups, and the Sis hen Pill control group (P <0. 05). Compared with the Sis hen Pill control group, there was statistical difference in all indices except CD45+ value in the low dose SWPSM group (P <0. 05). Compared with the low dose WJ R group, the expression of CD3+% decreased in high and middle dose WJR groups, and the Sis hen Pill control group; CD4+ /CD8+ increased in the Sishen Pill control group and the high dose SWPSM group (all P < 0. 05). CONCLUSIONS: WJR showed better treatment effect. The mechanism of Shen warming Pi strengthening method might be achieved by regulating expressions of CD45+% and CD3+%, and CD4+ /CD8+ ratios.

[Shen warming Pi strengthening method intervened IBS-D rats: an efficacy assessment].

OBJECTIVE: IBS-D rat model was established to assess the effect of Shen warming Pi strengthening method (SWPSM) for intervening diarrhea-predominant irritable bowel syndrome (IBS-D) by observing rats' general state, stool properties, AWR ranking, and histopathological changes. METHODS: Totally 72 rats were randomly divided into 6 groups, i.e. the normal group, the model group, the high, middle, low dose SWPSM groups, and the control group, 12 in each group. The IBS-D rat model was successfully established referring to AL-Chaer ED's modeling method. After modeling high, middle, and low dose SWPS Recipe boil-free granules were given by gastrogavage to rats in corresponding treatment groups. Sishen Pill boil-free granule was given by gastrogavage to those in the control group. Equal volume of normal saline was given by gastrogavage to rats in the model group. The medication lasted for 2 weeks. Rats' general state, stool properties, abdominal withdrawal reflex (AWR) ranking, and histopathological changes were observed. RESULTS: After treatment, the general state of all rats got im- provement to various degrees. The improvement in the high and middle dose SWPS Recipe groups were superior to that in the low dose SWPS Recipe group and the control group (P < 0.05). There was no statistical difference in the growth rate between after and before treatment in each group (P > 0.05). Compared with the model group and the low dose SWPS Recipe group, the defecation amount within 4 h was less in the high and middle dose SWPS Recipe groups and the control group (P < 0.05). The Bristol ranking score, average ranking of loose stool, ratio of dry stool and wet stool were lower in the high and middle dose SWPS Recipe groups than in the control group and the low dose SWPS Recipe group (P < 0.05). The AWR ranking score was lower in the high and middle dose SWPS Recipe groups than in the control group when the volume of balloon dilation was 1.5 mL. There was no organic change of histological or morphological observation. CONCLUSIONS: High sensitive IBS-D model was proved to be reliable. SWPSM could reduce the quantity of stools, lower Bristol ranking score, average ranking of loose stools as well as ratios of dry stool and wet stool, contributing to reducing the high sensitivity of rats' visceral organs to some extent.

Synthesis, characterization, and antitumor evaluation of the albumin-SN38 conjugate.

7-Ethyl-10-hydroxycamptothecin (SN38), the active metabolite of irinotecan, exerts a 100-fold to 1000-fold higher effect than irinotecan itself against several tumor cell lines. However, the water insolubility of SN38 has prevented its direct use as an antitumor drug in the clinic. To improve the water solubility and antitumor efficacy, SN38 was covalently attached to the only free sulfhydryl at cysteine-34 on the BSA site specifically through a thiol-binding linker to form a prodrug BSA-SN38 conjugate (BSA : SN38=1 : 1). The water solubility of this conjugate was similar to albumin using the current method. Also, SN38 loading in this conjugate became controllable. Size-exclusion chromatography purification and UV characterization of the SDS-PAGE electrophoresis product were carried out. Then, an MTT assay was carried out to test the antitumor effect of this conjugate on five colon cancer cell lines in vitro. The 72 h IC50 values of the BSA-SN38 conjugate ranged from 1.5 to 6.1 µmol/l. A colorectal peritoneal carcinomatosis model in mice was established to determine the intraperitoneal chemotherapy effect of the BSA-SN38 conjugate. The BSA-SN38 conjugate at an SN38 equivalent dose of 10 mg/kg/day was administrated every 4 days. Eighteen days after manipulation, the mice were euthanized and the tumors in the abdominal cavity were collected and weighed. Tumors in the BSA-SN38 conjugate treatment group (m=0.21 ± 0.15 g) were found to be significantly (P=5) lighter than those in the NS control group (m=4.74±0.73 g). The results indicated that this water-soluble BSA-SN38 conjugate exerted a strong antitumor effect on colorectal carcinoma.

Efficient inhibition of intraperitoneal ovarian cancer growth in nude mice by liposomal delivery of short hairpin RNA against STAT3.

AIM: Signal transducer and activator of transcription 3 (STAT3) plays an important role in the tumor formation, prognosis and chemoresistance of ovarian cancer. Our goal was to investigate the effect of silencing STAT3 on ovarian cancer cell apoptosis, proliferation, angiogenesis and expression of key targets in vitro and in vivo. METHODS: The ovarian cancer cell lines A2780CP and A2780s were used. STAT3 was knocked down by the plasmid-based short hairpin RNA (shRNA) expression system. In vitro, a colony formation assay and Hoechst staining were used to examine cell proliferation and apoptosis. The expression level of STAT3 and apoptosis-related proteins were determined by Western blot. The A2780CP intraperitoneal model was used to evaluate the effect of shSTAT3 on tumor growth in mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunostaining, respectively. RESULTS: Treatment with shSTAT3 resulted in apoptosis and inhibition of cell proliferation in vitro. Western blot analysis demonstrated that shSTAT3 induced the expression of cleaved caspase-3 and reduced the expression of survivin, Bcl-2 and vascular endothelial growth factor. In vivo, the tumor weight was reduced to 13.46% of 5% glucose by shSTAT3/lipoplexes (P < 0.01), accompanied with apoptosis induction (P < 0.01), proliferation inhibition (P < 0.01) and angiogenesis inhibition (P < 0.01). CONCLUSIONS: We find that treatment with shSTAT3 inhibits tumor growth in vitro and in vivo by inducing apoptosis and inhibiting cell proliferation. This work should provide the scientific foundation for future investigation of shSTAT3 as a strategy for ovarian cancer gene therapy and the combination of gene therapy with chemotherapy.

Curative effect of warming kidney and fortifying spleen recipe on diarrhea-predominant irritable bowel syndrome.

OBJECTIVE: To observe the curative effect of a recipe for warming the kidney and fortifying the spleen on diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS: This multi-center, double-blind, randomized, and controlled trial included 240 patients that met the inclusion criterion and were then divided into two groups of 120. Patients in the treatment group (group A) took modified Sishen Wan orally for warming the kidney and fortifying the spleen and patients in the control group (group B) took a placebo, Chao Maiya, for 4 weeks. 28 days after withdrawal, there was a 6-month follow-up to observe patient recurrence condition. The total effective rate, curative effect, and recurrence rate were evaluated after treatment. RESULTS: There was statistical difference (P < 0.01) between the two groups in total effective rate (92.24% in the treatment group and 49.07% in the control group), in curative effect of TCM syndrome (90.52% and 47.22%, respectively), and in the recurrence rate (15.79% and 56.86%, respectively) within 6 months after treatment. CONCLUSION: Modified Sishen Wan, for warming the kidney and fortifying the spleen, can effectively treat IBS-D and better control its recurrence.