Dihydrotanshinone I inhibits gallbladder cancer growth by targeting the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation.

BACKGROUND: Gallbladder cancer (GBC) poses a significant risk to human health. Its development is influenced by numerous factors, particularly the homeostasis of reactive oxygen species (ROS) within cells. This homeostasis is crucial for tumor cell survival, and abnormal regulation of ROS is associated with the occurrence and progression of many cancers. Dihydrotanshinone I (DHT I), a biologically effective ingredient isolated from Salvia miltiorrhiza, has exhibited cytotoxic properties against various tumor cells by inducing apoptosis. However, the precise molecular mechanisms by which dht I exerts its cytotoxic effects remain unclear. PURPOSE: To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms. METHODS: The proliferation of GBC cells, NOZ and SGC-996, was assessed using various assays, including CCK-8 assay, colony formation assay and EdU staining. We also examined cell apoptosis, cell cycle progression, ROS levels, and alterations in mitochondrial membrane potential to delve into the intricate molecular mechanism. Quantitative PCR (qPCR), immunofluorescence staining, and Western blotting were performed to evaluate target gene expression at both the mRNA and protein levels. The correlation between nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1) were examined using co-immunoprecipitation. Finally, the in vivo effect of dht I was investigated using a xenograft model of gallbladder cancer in mice. RESULTS: Our research findings indicated that dht I exerted cytotoxic effects on GBC cells, including inhibiting proliferation, disrupting mitochondrial membrane potential, inducing oxidative stress and apoptosis. Our in vivo studies substantiated the inhibition of dht I on tumor growth in xenograft nude mice. Mechanistically, dht I primarily targeted Nrf2 by promoting Keap1 mediated Nrf2 degradation and inhibiting protein kinase C (PKC) induced Nrf2 phosphorylation. This leads to the suppression of Nrf2 nuclear translocation and reduction of its target gene expression. Moreover, Nrf2 overexpression effectively counteracted the anti-tumor effects of dht I, while Nrf2 knockdown significantly enhanced the inhibitory effect of dht I on GBC. Meanwhile, PKC inhibitors and nuclear import inhibitors increased the sensitivity of GBC cells to dht I treatment. Conversely, Nrf2 activators, proteasome inhibitors, antioxidants and PKC activators all antagonized dht I induced apoptosis and ROS generation in NOZ and SGC-996 cells. CONCLUSION: Our findings indicated that dht I inhibited the growth of GBC cells by regulating the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation. These insights provide a strong rationale for further investigation of dht I as a potential therapeutic agent for GBC treatment.

Revealing the "Yin-Jing" mystery veil of Platycodon grandiflorum by potentiating therapeutic effects and lung-oriented guidance property.

ETHNIC PHARMACOLOGICAL RELEVANCE: "Yin-Jing" medicine (YJM) has been widely used by both ancient and modern Chinese medicine practitioners during long-term clinical practice. However, it remains unclear how to best guide other medicines to the targeted organs in a traditional Chinese medicine (TCM) prescription. Here, in an attempt to explain the scientific connotation of the YJM property (YJMP) attributed to a basic TCM theory, Platycodon grandiflorum (PG) was chosen as a case study to reveal the mystery of YJMP theory. AIM OF THE STUDY: The main purpose of this study is to employ modern chemical and molecular biology methods to confirm the "Yin-Jing" effect of PG, and further clarify its material basis and related possible mechanism. MATERIALS AND METHODS: The ammonia-induced lung injury rat model was utilized to determine the optimal dosage of traditional prescription Hui Yan Zhu Yu decoction (HYZYD) using Wright Giemsa staining, HE staining, Masson staining, and TUNEL analysis. With the same way, PG was confirmed to have potentiating therapeutic effect (PTE) by comparison with HYZYD and [HYZYD-PG]. TMT proteomics was used to reveal the "Yin-Jing" mechanism of action. Western blot assay (WB) was employed for verification of differentially expressed proteins. Additionally, four non-crossing fragmentations (Fr. A-D) were characterized by RPLC/SEC-ELSD and HILIC-ESI--Q-OT-IT-MS techniques. The PTE and guidance property assays were utilized to evaluate "Yin-Jing" functions by a compatible combination of hydroxysafflor yellow A (HYA) using qPCR, FCM, WB, HPLC, high content cell imaging (HCI) and high-resolution live-cell imaging (HRLCI) techniques. RESULTS: The HYZYD-M (medium dose group) significantly improved the lung injury level in a pneumonia model of rats. PG enhanced the therapeutic effect of HYZYD ascribed to Yin-Jing PTE functions. TMT proteomics revealed a category of differentially expressed proteins ascribed to Golgi-ER between HYZYD and [HYZYD-PG]. Fr. C (i.e., saponins) and Fr. D (i.e., lipids) were determined as therapeutic fragmentations via the LPS-induced A549 cell injury model; however, Fr. B (fructooligosaccharides and small Mw fructans) had no therapeutic effect. Further compatibility PTE assays confirmed Fr. B significantly improved efficiency by a combination of HYA. The guidance assays showed Fr. B could significantly increase the uptake and distribution of HYA into lung cells and tissues. HCI assays showed that Fr. B increased uptake of HYA accompanied by significant activation of Golgi-ER. Unlike Fr. B, HRLCI showed that Fr. A, C and D were not only unobvious activations of Golgi-ER but also insignificant facilitation of colocalizations between HYA and Golgi-ER. CONCLUSIONS: Fr. B is believed to be a key YJMP material basis of PG attributed to Yin-Jing PTE with characteristic of lung-oriented guidance property, whereas another abound Fr. C was determined to have synergistic effects rather than Yin-Jing material basis.

Risk of venous thromboembolism in children and adolescents with inflammatory bowel disease: A systematic review and meta-analysis.

BACKGROUND: A two- to three-fold increased risk of venous thrombotic events (VTE) has been demonstrated in patients with inflammatory bowel disease (IBD) compared to the general population, but less is known about the risk of VTE in child- and pediatric-onset IBD. In recent years, several studies have reported the rising incidence rate of VTE in juvenile patients with IBD, and the related risk factors have been explored. AIM: To evaluate the risk of VTE in children and adolescents with IBD. METHODS: Articles published up to April 2021 were retrieved from PubMed, Embase, Cochrane Library, Web of Science, SinoMed, CNKI, and WANFANG. Data from observational studies and clinical work were extracted. The outcome was the occurrence of VTE according to the type of IBD. The available odds ratio (OR) and the corresponding 95% confidence interval (CI) were extracted to compare the outcomes. Effect size (P), odds ratio (OR), and 95%CI were used to assess the association between VTE risk and IBD disease. Subgroup analyses stratified by subtypes of VTE and IBD were performed. RESULTS: Twelve studies (7450272 IBD patients) were included in the meta-analysis. Child and adolescent IBD patients showed increased VTE risk (P = 0.02, 95%CI: 0.01-0.03). Subgroup analyses stratified by IBD (ulcerative colitis (UC): P = 0.05, 95%CI: 0.03-0.06; Crohn's disease (CD): P = 0.02, 95%CI: 0.00-0.04) and VTE subtypes (portal vein thrombosis: P = 0.04, 95%CI: 0.02-0.06; deep vein thrombosis: P = 0.03, 95%CI: 0.01-0.05; central venous catheter-related thrombosis: P = 0.23, 95%CI: 0.00-0.46; thromboembolic events: P = 0.02, 95%CI: 0.01-0.03) revealed a significant correlation between VTE risk and IBD. Patients with IBD were more susceptible to VTE risk than those without IBD (OR = 2.99, 95%CI: 1.45-6.18). The funnel plot was asymmetric, suggesting the presence of significant publication bias. Pediatric and adolescent IBD patients have an increased VTE risk. UC and CD patients exhibited a high risk of VTE. The risk of VTE subtypes was increased in IBD patients. CONCLUSION: The current meta-analysis showed that the incidence and risk of VTE are significantly increased in pediatric and adolescent IBD patients. Thus, IBD might be a risk factor for VTE in children and young adults. High-quality prospective cohort studies are necessary to confirm these findings.

FGF21 Enhances Therapeutic Efficacy and Reduces Side Effects of Dexamethasone in Treatment of Rheumatoid Arthritis.

In order to investigate efficacy of FGF21 combine dexamethasone (Dex) on rheumatoid arthritis (RA) meanwhile reduce side effects of dexamethasone. We used combination therapy (Dex 15 mg/kg + FGF21 0.25 mg/kg, Dex 15 mg/kg + FGF21 0.5 mg/kg or Dex 15 mg/kg + FGF21 1 mg/kg) and monotherapy (Dex 15 mg/kg or FGF21 1 mg/kg) to treat CIA mice induced by chicken type II collagen, respectively. The effects of treatment were determined by arthritis severity score, histological damage, and cytokine production. The levels of oxidative stress parameters, liver functions, and other blood biochemical indexes were detected to determine FGF21 efficiency to side effects of dexamethasone. Oil red O was performed to detect the effects of FGF21 and dexamethasone on fat accumulation in HepG2 cells. The mechanism of FGF21 improves the side effects of dexamethasone which was analyzed by Western blotting. This combination proved to be therapeutically more effective than dexamethasone or FGF21 used singly. FGF21 regulates oxidative stress and lipid metabolism by upregulating dexamethasone-inhibited SIRT-1 and then activating downstream Nrf-2/HO-1and PGC-1. FGF21 and dexamethasone are highly effective in the treatment of arthritis; meanwhile, FGF21 may overcome the limited therapeutic response and Cushing syndrome associated with dexamethasone.

Interleukin-22 receptor 1 is expressed in multinucleated giant cells: A study on intestinal tuberculosis and Crohn's disease.

BACKGROUND: It is challenging to distinguish intestinal tuberculosis from Crohn's disease due to dynamic changes in epidemiology and similar clinical characteristics. Recent studies have shown that polymorphisms in genes involved in the interleukin (IL)-23/IL-17 axis may affect intestinal mucosal immunity by affecting the differentiation of Th17 cells. AIM: To investigate the specific single-nucleotide polymorphisms (SNPs) in genes involved in the IL-23/IL-17 axis and possible pathways that affect susceptibility to intestinal tuberculosis and Crohn's disease. METHODS: We analysed 133 patients with intestinal tuberculosis, 128 with Crohn's disease, and 500 normal controls. DNA was extracted from paraffin-embedded specimens or whole blood. Four SNPs in the IL23/Th17 axis (IL22 rs2227473, IL1ß rs1143627, TGFß rs4803455, and IL17 rs8193036) were genotyped with TaqMan assays. The transcriptional activity levels of different genotypes of rs2227473 were detected by dual luciferase reporter gene assay. The expression of IL-22R1 in different intestinal diseases was detected by immunohistochemistry. RESULTS: The A allele frequency of rs2227473 (P = 0.030, odds ratio = 0.60, 95% confidence interval: 0.37-0.95) showed an abnormal distribution between intestinal tuberculosis and healthy controls. The presence of the A allele was associated with a higher IL-22 transcriptional activity (P < 0.05). In addition, IL-22R1 was expressed in intestinal lymphoid tissues, especially under conditions of intestinal tuberculosis, and highly expressed in macrophage-derived Langhans giant cells. The results of immunohistochemistry showed that the expression of IL-22R1 in patients with Crohn's disease and intestinal tuberculosis was significantly higher than that in patients with intestinal polyps and colon cancer (P < 0.01). CONCLUSION: High IL-22 expression seems to be a protective factor for intestinal tuberculosis. IL-22R1 is expressed in Langhans giant cells, suggesting that the IL-22/IL-22R1 system links adaptive and innate immunity.

The Polymorphism rs17525495 of LTA4H Is Associated with Susceptibility of Crohn's Disease instead of Intestinal Tuberculosis in a Chinese Han Population.

BACKGROUND: Because of the similarity of intestinal tuberculosis and Crohn's disease in disease phenotype, differential diagnosis has always been a clinical problem. Arachidonic acid metabolites play an important role in the inflammatory response of intestinal tuberculosis and Crohn's disease. Recent studies have shown that the polymorphism locus in the promoter region of LTA4H gene affects LTB4 expression level and the susceptibility to extrapulmonary tuberculosis. Thus, we identified a total of 148 patients with intestinal tuberculosis, 145 with Crohn's disease, and 700 normal controls in this study. METHODS: All the study participants were local Han people from Jiangxi Province in the past eleven years. DNA was extracted from the paraffin-embedded specimens or the whole blood. The LTA4H promoter SNP (rs17525495) was genotyped with TaqMan assay. RESULTS: The T-alleles frequency was not significantly increased in patients with intestinal tuberculosis compared with healthy control group (p=0.630; OR=1.07; 95%CI=0.81-1.41), while patients with Crohn's disease have significantly increased T allele frequency compared with healthy population (p=0.032; OR=1.34; 95%CI=1.03-1.75). During treatment, the presence of the T allele significantly increased the proportion of Crohn's patients requiring glucocorticoids (p<0.05). CONCLUSIONS: The T allele of LTA4H gene SNP (rs17525495) is a risk factor for Crohn's disease instead of intestinal tuberculosis. More importantly, there may be a potential association of the different genotypes of rs17525495 with the treatment efficacy of 5-ASA and glucocorticoids in patients with Crohn's disease. The association between LTA4H polymorphism and drugs therapeutic effects might contribute to the practice of precision medicine and the prediction of clinical outcomes.

Improving hyperglycemic effect of FGF-21 is associated with alleviating inflammatory state in diabetes.

Type 2 diabetes mellitus (T2DM) is accompanied by abnormal glucose metabolism and low-grade chronic inflammation. Fibroblast growth factor 21 (FGF-21) is a novel metabolic regulator and can function as an endocrine hormone to regulate glucose and lipid metabolism. Recently, FGF-21 was found to have anti-inflammatory effect, to our knowledge, the effect of FGF-21 on inflammatory state in diabetes has not been elucidated. Here, we use db/db mice as a Type 2 diabetes model to determine whether FGF-21 alleviates inflammatory state while improves hyperglycemia. Our results demonstrated that FGF-21 not only showed potent long lasting hypoglycemic effect, but also demonstrated strong anti-inflammatory effect in the serum and white adipose tissue. Besides, in vitro experiments, insulin resistance (IR) was induced in 3T3-L1 adipocytes by treating with TNF-α. Our results showed that TNF-α impaired glucose metabolism of 3T3-L1 adipocytes but FGF-21 repressed gene expression of inflammatory factors caused by IR and consequently improved the glucose metabolism in 3T3-L1 adipocytes. Furthermore, FGF-21 ameliorated glucose uptake of TNF-α-induced IR in 3T3-L1 adipocytes by inhibiting NF-κB signaling pathway.

FGF-21 Plays a Crucial Role in the Glucose Uptake of Activated Monocytes.

Monocytes display a gradual change in metabolism during inflammation. When activated, the increase in glucose utilization is important for monocytes to participate in immune and inflammatory responses. Further studies on the mechanism underlying this biological phenomenon may provide a new understanding of the relationship between immune response and metabolism. The THP-1 cells were used as a monocyte model. The cells were activated with lipopolysaccharide (LPS). Glucose uptake was measured using flow cytometry. The expression of fibroblast growth factor 21 (FGF-21), glucose transporter 1 (GLUT-1), and other FGF-21 signaling pathway-related factor mRNAs was determined by real-time polymerase chain reaction. Further, the relationship between FGF-21 expression in monocytes and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) signaling pathway was determined by Western blotting. LPS elevated FGF-21 expression in monocytic THP-1 cells in vitro. Functional assays showed that the phenomenon in which LPS and FGF-21 stimulated glucose uptake in monocytic THP-1 cells could be inhibited by FGFR inhibitor. The mechanism of elevation of FGF-21 was found to involve the PI3K/Akt signaling pathway. This study indicated that FGF-21 could regulate the immune response indirectly by influencing the glucose uptake of activated monocytes cells.

Dryocrassin ABBA Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells Through a Caspase-Dependent Mitochondrial Pathway.

BACKGROUND: Biological and pharmacological activities of dryocrassin ABBA, a phloroglucinol derivative extracted from Dryopteris crassirhizoma, have attracted attention. In this study, the apoptotic effect of dryocrassin ABBA on human hepatocellular carcinoma HepG2 cells was investigated. MATERIALS AND METHODS: We tested the effects of dryocrassin ABBA on HepG2 in vitro by MTT, flow cytometry, real-time PCR, and Western blotting. KM male mice were used to detect the effect of dryocrassin ABBA on H22 cells in vivo. RESULTS: Dryocrassin ABBA inhibited the growth of HepG2 cells in a concentration-dependent manner. After treatment with 25, 50, and 75 µg/mL dryocrassin ABBA, the cell viability was 68%, 60% and 49%, respectively. Dryocrassin ABBA was able to induce apoptosis, measured by propidium iodide (PI)/annexin V-FITC double staining. The results of real-time PCR and Western ting showed that dryocrassin ABBA up-regulated p53 and Bax expression and inhibited Bcl-2 expression which led to an activation of caspase-3 and caspase-7 in the cytosol, and then induction of cell apoptosis. In vivo experiments also showed that dryocrassin ABBA treatment significantly suppressed tumor growth, without major side effects. CONCLUSIONS: Overall, these findings provide evidence that dryocrassin ABBA may induce apoptosis in human hepatocellular carcinoma cells through a caspase-mediated mitochondrial pathway.

Treatment of CIA Mice with FGF21 Down-regulates TH17-IL-17 Axis.

Recently, FGF21 was reported to play an important role in anti-inflammation. The aim of the study is to explore the mechanism for FGF21 alleviating inflammation of CIA. CIA mice were injected with FGF21 once a day for 28 days after first booster immunization. The results showed that FGF21 alleviates arthritis severity and decreases serum anti-CII antibodies levels in CIA mice. Compared with CIA model, the number of the splenic TH17 cells was significantly decreased in FGF21-treated mice. FGF21 treatment reduced the mRNA expression of IL-17, TNF-α, IL-1ß, IL-6, IL-8, and MMP3 and increased level of IL-10 in the spleen tissue. The expression of STAT3 and phosphorylated STAT3 was suppressed in FGF21-treated group. The mRNA expression of RORγt and IL-23 also decreased. In conclusion, these findings suggest that the beneficial effects of FGF21 on CIA mice were achieved by down-regulating Th17-IL-17 axis through STAT3/RORγt pathway. Modulating of Th17-mediated inflammatory response may be one of the mechanisms for FGF21 attenuating inflammation in CIA.

[The synergistic effect of FGF-21 and insulin on regulating glucose metabolism and its mechanism].

Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.

[FGF-21 protects H9c2 cardiomyoblasts against hydrogen peroxide-induced oxidative stress injury].

Fibroblast growth factor-21 (FGF-21) is an important metabolism regulator, however, whether FGF-21 has effects on cardiovascular remains unclear. In this study, H2O2-induced injury in H9c2 cells was used as a cell model, the anti-apoptosis potential and mechanism of FGF-21 against oxidative injury were evaluated by MTT assay, flow cytometry assay and real-time PCR. The results showed that FGF-21 could increase the cell survival of H2O2-induced injury in H9c2 cells and prevent H9c2 cells from oxidative stress-induced apoptosis. Furthermore, FGF-21 can elevate SOD activity and regulate Bcl-2/Bax expression in H9c2 cells. The results suggest that FGF-21 have protective effect against the H2O2-induced apoptosis in H9c2 cells.

[Therapeutic efficacy of three bispecific antibodies on rheumatoid arthritis mice models].

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.

Krüppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer.

AIM: To investigate Krüppel-like factor 8 (KLF8) expression in gastric cancer and its relationship with angiogenesis and prognosis of gastric cancer. METHODS: One hundred and fifty-four patients with gastric cancer who underwent successful curative resection were retrospectively enrolled in the study. Fifty tumor-adjacent healthy gastric tissues (≥ 5 cm from the tumor margin) obtained during the original resection were randomly selected for comparative analysis. In situ expression of KLF8 and CD34 proteins were examined by immunohistochemistry. The intratumoral microvessel density (MVD) was determined by manually counting the immunostained CD34-positive endothelial cells in three consecutive high-magnification fields (× 200). The relationship between differential KLF8 expression and MVD was assessed using Spearman's correlation coefficient test. χ² test was performed to evaluate the effects of differential KLF8 expression on clinicopathologic factors. Kaplan-Meier and multivariate Cox survival analyses were used to assess the prognostic value of differential KLF8 expression in gastric cancer. RESULTS: Significantly higher levels of KLF8 protein were detected in gastric cancer tissues than in the adjacent non-cancerous tissues (54.5% vs 34.0%, P < 0.05). KLF8 expression was associated with tumor size (P < 0.001), local invasion (P = 0.005), regional lymph node metastasis (P = 0.029), distant metastasis (P = 0.023), and tumor node metastasis (TNM) stage (P = 0.002), as well as the MVD (r = 0.392, P < 0.001). Patients with KLF8 positive expression had poorer overall survival (P < 0.001) and cancer-specific survival (P < 0.001) than those with negative expression. Multivariate analysis demonstrated that KLF8 expression independently affected both overall and cancer-specific survival of gastric cancer patients (P = 0.035 and 0.042, respectively). CONCLUSION: KLF8 is closely associated with gastric tumor progression, angiogenesis and poor prognosis, suggesting it may represent a novel prognostic biomarker and therapeutic target for gastric cancer.

[Optimization and characterization of a novel FGF21 mutant].

Fibroblast growth factor 21 (FGF21) is a member of FGF family. It has been demonstrated that FGF21 is an independent, safe and effective regulator of blood glucose levels in vivo. In order to improve the activity of FGF21, we exchanged the beta10-beta12 domain of the human FGF21 with that of the mouse FGF21 to construct a novel FGF21 gene (named hmFGF21), and then subcloned hmFGF21 gene into the SUMO expression vector to create pSUMO-hmFGF21 and transformed it into E. coli Rosetta for expression of the fusion protein SUMO-hmFGF21. Both in vitro and in vivo glucose regulation activity of hmFGF21 was evaluated. The SDS-PAGE result showed that compared with wild-type hFGF21, the soluble expression of hmFGF21 increased about 2-fold. HmFGF21 was more potent in stimulation of glucose uptake in HepG2 cells in vitro. The results of anti-diabetic effect on db/db mice demonstrated that hmFGF21 had better efficacy on controlling the blood glucose of the db/db diabetic animals than wild-type hFGF21. These results suggest that the biological properties of FGF21 are significantly improved by optimization.

[Affinity optimization of an anti-hIL17A single-chain Fv antibody through error-prone PCR technique].

AIM: To optimize antibody affinity of a humanized anti-hIL17A single-chain Fv (scFV) antibody through error-prone polymerase chain reaction (PCR) so as to develop a therapeutic humanized antibody for the treatment of rheumatoid arthritis (RA). METHODS: Variable regions of heavy chain and light chain were subjected to random mutation by error-prone PCR and a scFv library was constructed by overlapping PCR. The library was screened for improved mutants by the bacterial inner membrane display technique and flow cytometry (FCM). Real-time PCR was used to detect neutralization effects of the purified scFv antibody mutants on mRNA expressions of IL-6 and IL-8 in HeLa cells stimulated by hIL17A. RESULTS: We obtained five mutants with improved affinity. FCM revealed that the affinity of three clones was greatly enhanced as compared with the parent clone. All mutants retained binding specificity to hIL17A. Real-time PCR results showed that all five mutants could block hIL17A stimulation of HeLa cells to express IL-6 and IL-8, and the neutralization effects were positively related to mutant affinity. CONCLUSION: Error-prone PCR technique is a feasible method for antibody affinity optimization in vitro, which is able to improve the affinity and neutralization capacity of antibody on the basis of its unchanged antigen binding specificity. This study provides potential drug candidates aimed for the treatment of rheumatoid arthritis and an alternative method of optimizing antibody affinity in vitro.

[Establishment of a novel cell model targeted on FGF-21 receptor for screening anti-diabetic drug candidates].

The aim of this project is to establish a fibroblast growth factor-21 (FGF-21) signaling pathway targeted cell model, for screening a class of FGF-21 receptor agonists as anti-diabetic candidates. FGF-21 requires beta klotho transmembrane proteins as co-receptor for the activation of tyrosine kinase FGF receptor (FGFR) signaling, thereby activating a series of intracellular signaling pathways and regulating gene transcription for glucose metabolism. Firstly a recombinant plasmid expressing co-receptor beta klotho and EGFP reporter genes was constructed. After introducing the recombinant plasmid into package cells, the cell culture supernatant was used to infect 3T3-L1 cells, which were then screened for stably expressing beta klotho gene. Administration of FGF-21 increased the expression of GLUT1 and stimulated GLUT1-mediated glucose uptake. This novel cell model can be conveniently used in high-throughput drug screening of FGF-21 or FGF-21 analogues.