Gold nanoparticles-conjugation of irisin enhances therapeutic effect by improving cardiac function and attenuating inflammation in sepsis.

Irisin is considered to be a promising therapeutic approach for cardiac depression and inflammatory disorders. The short half-life of irisin impeded its use and drug efficacy in the treatment. This study aimed to examine if pegylated gold nanoparticles-conjugated to irisin would improve therapeutic effects in cecal ligation and puncture (CLP)-induced sepsis in mice. Recombinant irisin were conjugated to a pegylated gold nanoparticle, which was given to mice exposed to CLP. The cecal ligation procedure and sham on mice were operated and assigned to one of following five groups: (I) CLP group: The mouse models underwent the CLP surgical procedure and received only vehicle saline treatment (n = 5); (II) CLP + soluble Irisin: The mouse underwent the CLP and received an intramuscular injection (i.m) (TA) injection of 1 ug of soluble irisin into each tibialis anterior (TA) leg (n = 5); (III) CLP + Gold nanoparticle-conjugated to Irisin: The mouse models underwent the CLP and received an i.m (TA) injection of 1 µg of Gold nanoparticle-irisin via intramuscular injection (TA) into each leg (n = 5); (IV) CLP + Gold nanoparticles- conjugated to IgG: The mouse underwent the CLP and received an i.m (TA) injection of gold nanoparticles conjugated to IgG (n = 5). (V) Sham: The mouse underwent the surgical operation without conducting the CLP (n = 10). The post-operated animals were observed for one week, and survival rates were estimated. Echocardiography was performed to measure cardiac function at 12 h following CLP. TUNEL was employed to detect apoptosis in both cardiac and skeletal muscles; histology was conducted to assess tissue injury in muscles. Enzyme linked immunosorbent assay (ELISA) was conducted to examine release of interleukin 6 (IL6) and the tumor necrosis factor (TNF) alpha. Compared to the CLP control, soluble irisin treatment improved cardiac function recovery, as indicated by the fractional shortening (FS) and ejection fraction (EF). Irisin treatment exhibited reduced IL6 and TNF-alpha release in association with less apoptosis, lower muscle injury index and improved survival post-CLP. However, compared to soluble irisin treatment, gold nanoparticles-conjugated to irisin showed a significant improvement in cardiac function, suppression of apoptosis, reduced IL6 and TNF-alpha releases, decreased muscle injury and an improved survival rate of post-CLP. This study reveals that gold nanoparticles-conjugated irisin can serve to improve irisin's therapeutic effects over a longer course of treatment.

Irisin deficiency exacerbates diet-induced insulin resistance and cardiac dysfunction in type II diabetes in mice.

Irisin is involved in the regulation of a variety of physiological conditions, metabolism, and survival. We and others have demonstrated that irisin contributes critically to modulation of insulin resistance and the improvement of cardiac function. However, whether the deletion of irisin will regulate cardiac function and insulin sensitivity in type II diabetes remains unclear. We utilized the CRISPR/Cas-9 genome-editing system to delete irisin globally in mice and high-fat diet (HFD)-induced type II diabetes model. We found that irisin deficiency did not result in developmental abnormality during the adult stage, which illustrates normal cardiac function and insulin sensitivity assessed by glucose tolerance test in the absence of stress. The ultrastructural analysis of the transmission electronic microscope (TEM) indicated that deletion of irisin did not change the morphology of mitochondria in myocardium. Gene expression profiling showed that several key signaling pathways related to integrin signaling, extracellular matrix, and insulin-like growth factors signaling were coordinately downregulated by deletion of irisin. However, when mice were fed a high-fat diet and chow food for 16 wk, ablation of irisin in mice exposed to HFD resulted in much more severe insulin resistance, metabolic derangements, profound cardiac dysfunction, and hypertrophic response and remodeling as compared with wild-type control mice. Taken together, our results indicate that the loss of irisin exacerbates insulin resistance, metabolic disorders, and cardiac dysfunction in response to HFD and promotes myocardial remodeling and hypertrophic response. This evidence reveals the molecular evidence and the critical role of irisin in modulating insulin resistance and cardiac function in type II diabetes.NEW & NOTEWORTHY By utilizing the CRISPR/Cas-9 genome-editing system and high-fat diet (HFD)-induced type II diabetes model, our results provide direct evidence showing that the loss of irisin exacerbates cardiac dysfunction and insulin resistance while promoting myocardial remodeling and a hypertrophic response in HFD-induced diabetes. This study provides new insight into understanding the molecular evidence and the critical role of irisin in modulating insulin resistance and cardiac function in type II diabetes.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Inhibition of integrin alpha v/beta 5 mitigates the protective effect induced by irisin in hemorrhage.

INTRODUCTION: Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvß5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvß5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvß5 contributes to the effects of irisin during the hemorrhagic response. METHODS: Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35-45 mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5  µg/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin αvß5, or control RGDS (1 mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin ß5 with nanoparticle delivery of integrin ß5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues. RESULTS: Hemorrhage induced reduction of integrin αvß5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin ß5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin ß5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin αvß5, or knockdown of integrin ß5. CONCLUSION: Integrin αvß5 plays an important role for irisin in modulating the protective effect during hemorrhage.

Ultrawide-range radiation detection based on dynamic identification and analysis of the response of a monolithic active pixel sensor.

In this paper, we present an ultrawide-range radiation detection method based on dynamic recognition and analysis of the response signal of a monolithic active pixel sensor (MAPS). Our analysis of the MAPS response mechanism determined that adaptive adjustment of the sensor's integral time is key to quantification of ionizing radiation in an ultrawide range. We also determined that different data processing methods are required for accurate quantification of high and low radiation dose rates. The results of experiments conducted after calibration demonstrate that our technique is capable of radiation detection across five orders of magnitude (ranging from milligrays per hour to hundreds of grays per hour), with errors of less than 5%. Chip-based nuclear radiation detection can be realized using our technique, enabling MAPS to be used as a supplement to traditional detectors in characterization of unknown and complex radiation environments.

Quadruped Gait and Regulation of Apoptotic Factors in Tibiofemoral Joints following Intra-Articular rhPRG4 Injection in Prg4 Null Mice.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4-PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4-/- (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4.

[Clinical effect of laparoscopic transcystic drainage combined with common bile duct exploration for the patients with difficult biliary stones].

OBJECTIVE: To explore the feasibility and efficacy of laparoscopic transcystic drainage and common bile duct exploration in the treatment of patients with difficult biliary stones. METHODS: Between April 2020 and December 2021, eighteen patients with difficult biliary stones received laparoscopic transcystic drainage (C-tube technique) and common bile duct exploration. The clinical characteristics and outcomes were retrospectively collected. The safety and effectiveness of laparoscopic transcystic drainage and common bile duct exploration were analyzed. RESULTS: Among the eighteen patients with difficult biliary stones, thirteen patients received traditional laparoscopic transcystic drainage, and the remaining five received modified laparoscopic transcystic drainage. The mean surgical duration were (161±59) min (82-279 min), no bile duct stenosis or residual stone was observed in the patients receiving postoperative cholangiography via C-tube. The maximum volume of C-tube drainage was (500±163) mL/d (180-820 mL/d). Excluding three patients with early dislodgement of C-tube, among the fifteen patients with C-tube maintained, the median time of C-tube removal was 8 d (5-12 d). The duration of hospital stay was (12±3) d (7-21 d) for the 18 patients. Five C-tube related adverse events were observed, all of which occurred in the patients with traditional laparoscopic transcystic drainage, including two abnormal position of the C-tube, and three early dislocation of the C-tube. All the 5 adverse events caused no complications. Only one grade one complication occurred, which was in a patient with modified laparoscopic transcystic drainage. The patient demonstrated transient fever after C-tube removal, but there was no bile in the drainage tube and the subsequent CT examination confirmed no bile leakage. The fever spontaneously relieved with conservative observation, and the patient recovered uneventfully with discharge the next day. All the 18 patients were followed up for 1-20 months (median: 9 months). Normal liver function and no recurrence of stone were detected with ultrasonography or magnetic resonance cholangiopancreatography (MRCP). CONCLUSION: Laparoscopic transcystic drainage combined with common bile duct exploration is safe and feasible in the treatment of patients with difficult biliary stones. The short-term effect is good. Modified laparoscopic transcystic drainage approach may reduce the incidence of C-tube dislocation and bile leak.

The Essential Role of PRAK in Preserving Cardiac Function and Insulin Resistance in High-Fat Diet-Induced Diabetes.

Regulated/activated protein kinase (PRAK) plays a crucial role in modulating biological function. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high-fat diet (HFD). Wild-type and PRAK-/- mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance tests and insulin tolerance tests were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart. Western blot was employed to determine cellular signaling pathway. HFD-induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared with wild-type littermates. As compared with wild-type mice, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high-fat diet intervention. High-fat diet intervention displayed a decline in fractional shortening and ejection fraction. However, deletion of PRAK exacerbated the decline in cardiac function as compared with wild-type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and ßMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared with wild-type controls. Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.

[Research on quantitative method and contamination level of Salmonella enterica in raw pork from farmer's markets in Chengdu].

To optimize the quantitative detection method for Salmonella enterica and analyze the quantitative contamination level of Salmonella enterica in raw pork samples from farmer's markets in Chengdu. Based on qualitative detection standard method of Salmonella enterica in China (GB 4789.4-2016) and the quantitative detection method of FSIS in the United States (MLG 4.08 and MLG appendix 2.05 MPN), the selective enrichment broth, screening plate, identification method and quantitative dilution ratio in quantitative detection of Salmonella enterica were optimized using 70 samples of raw pork. The optimized method compared by student's t-test was used to detect 40 samples of raw pork collected from farmer's markets in Chengdu from June to October 2020. For isolation of Salmonella from raw pork samples, the coincidence degree of TTB enrichment solution was significantly higher than that of RV enrichment solution (0.93±0.32 vs 0.35±0.62,t=8.324,P=0.001) and the consistency of suspicious colonies screened by XLT4 plate was significantly higher than that of Salmonella chromogenic medium (0.77±0.09 vs 1.00±0.00,t=2.971,P =0.017). The MPN method used 4 successive gradient dilutions, namely 12 tube method, could obtain more accurate quantitative value for Salmonella enterica. The combined use of selective enrichment broth TTB, XLT4 plate, Real-time PCR and MALDI-TOF mass spectrometry could get better results for screening and identifying Salmonella enterica. The detection rate for Salmonella enterica isolated from raw pork in farmer's markets was 92.5% (37/40). The most of the Salmonella positive samples (83.8%, 31/37) were detected with a contamination level ranged from 0.1 to 55 MPN/g. The optimized quantitative detection method for Salmonella enterica in raw pork in this study can effectively screen the target bacteria and obtain more accurate quantitative value.

[Early efficacy of islet transplantation in the treatment of adult advanced diabetes].

Objective: To evaluate the safety and efficacy of islet transplantation for patients with advanced diabetes. Methods: Five cases of islet allotransplantation were performed on 4 adult recipients. The same blood type adult brain-dead pancreas donors were selected and the islets were prepared in GMP laboratory. The prepared islet suspension was slowly injected into the liver of the recipients within 30-60 minutes. The immunosuppressive regimen was a combination of basiliximab, tacrolimus and mycophenolate mofetil and TNF-alpha monoclonal antibody was used to reduce the post-transplant inflammatory response. Insulin was temporarily applied to control blood glucose after surgery, and the dosage of insulin was adjusted to decrease according to the blood glucose level until it was discontinued. Results: A total of 5 islet transplants were performed in 4 patients, including 1 patient who received the second islet transplantations. All operations were succeed and the blood glucose and portal pressure were stable during the operation. Exogenous insulin was continued to keep blood glucose level stable (4-12 mmol/L) after surgery. Four cases (including the one who received two islet transplantation) started to stop using insulin at the third to fourth week, and the insulin dosage of the other case was 74% lower than that before the operation, and no hypoglycemic reaction occurred in all patients after islet transplantation. The C-peptide level in 3 patients reached the normal range, and the level in one patient with type I diabetes (without insulin release) remained at 0.45-0.6 µg/L (0.15-0.2 nmol/L). In addition, one patient showed a rise in blood glucose again and continued to use insulin half a year after insulin discontinuation. Then, he was performed the second islet transplantation which showed good effect and stopped taking insulin in 10 days after surgery. There were 3 cases of liver puncture bleeding after opeation, of which 2 cases were treated with ultrasound radiofrequency ablation to stop bleeding, 1 case stopped spontaneously, and no other complications were found. Conclusions: Islet transplantation is effective in the treatment of advanced diabetes patients with small trauma and high safety, which is worthy of more promotion. Long-term efficacy and maintenance therapy still need further investigation.

[Risk factors for the development of liver cancer in patients with hepatitis B-related liver cirrhosis treated with long-term nucleos(t)ide analogues].

Objective: To retrospectively analyze the risk factors for the development of liver cancer in patients with hepatitis B-related liver cirrhosis (LC) treated and fully managed with long-term nucleos(t)ide analogues (NAs). Methods: The study subjects were derived from the follow-up cohort of chronic hepatitis B and liver cirrhosis who received antiviral therapy in the Department of Infectious Diseases of the First Affiliated Hospital of Guangxi Medical University from February 2004 to September 2019. LC patients who met the inclusion criteria were enrolled. The life-table method was used to calculate the incidence of liver cancer. Multivariable Cox regression model was used to analyze the risk factors that may affect the development of liver cancer in patients with LC. A subgroup analysis was conducted in liver cirrhotic patients who developed liver cancer to evaluate the effectiveness of antiviral treatment compliance. The (2) test was used for rate comparison. Results: The median follow-up time of 198 LC cases treated with NAs was 6.0 years (1.0-15.3 years). By the end of the visit: (1) 16.2% (32/198) of LC patients had developed liver cancer, and the cumulative incidence of liver cancer in 1, 3, 5, 7, and 9 years were 0, 8.9%, 14.3%, 18.6%, and 23.4%, respectively, with an average annual incidence of 3.1%. Among the 32 cases with liver cancer, 68.7% had developed small liver cancer (22/32). (2) Univariate Cox model analysis showed that the development of liver cancer was related to four factors, i.e., the presence or absence of LC nodules, whether the baseline was first-line medication, the family history of liver cancer, and patient compliance. The results of multivariate Cox model analysis showed that poor patient compliance and baseline non-first-line medication were risk factors for liver cancer. (3) The results of log-rank test subgroup analysis showed that the 5-year cumulative incidence of liver cancer in patients with hardened nodules was significantly higher than that of patients without hardened nodules (21.7% vs. 11.5%, P = 0.029). The 5-year cumulative incidence of liver cancer in patients with non-first-line drugs was significantly higher than that of patients with first-line drugs (22.0% vs.8.2%, P = 0.003). The 5-year cumulative incidence of liver cancer in patients with poor compliance was significantly higher than that of patients with good compliance (21.3% vs. 12.7%, P = 0.014). The 5-year cumulative incidence of liver cancer in patients with a family history of liver cancer was significantly higher than that of patients without a family history of liver cancer (22.3% vs. 8.1%, P = 0.006). (4) Compared with patients with poor compliance, patients with good compliance had higher HBV DNA negative serconversion rate (98.7% vs. 87.8%, P = 0.005), and a lower virological breakthrough rate (12.1% vs. 29.3%, P = 0.007). Conclusion: The long-term NAs antiviral therapy can reduce the risk of liver cancer, but it cannot completely prevent the development of liver cancer, especially in patients with a family history of liver cancer and baseline hardened nodules (high risk of liver cancer). Furthermore, the complete management can improve patient compliance, ensure the efficacy of antiviral therapy, and reduce the risk of liver cancer development, so to achieve secondary prevention of liver cancer, i.e., early detection, diagnosis and treatment.

[Liver histological status and clinic outcome in HBeAg-negative chronic hepatitis B with low viral load].

Objective: To retrospectively analyze the serological, virological, biochemical, liver histological status and clinical outcomes in HBeAg-negative chronic hepatitis B (CHB) patients with low HBV viral load, and to explore the necessity of antiviral therapy for these patients. Methods: A total of 99 HBeAg-negative CHB patients with HBV DNA level < 4 lg copies/ml who performed liver biopsy at the baseline were enrolled from the follow-up cohort. Among them, 23 cases received the second liver biopsy during follow-up. The relationships among the degree of inflammation and fibrosis of liver tissues, the status of HBsAg and HBcAg, age, gender, family history, HBV DNA load, serological markers and other indicators were analyzed. The pathological differences between two liver biopsy examinations were compared. The effect of nucleos(t)ide analogues (NAs) treatment on patient's clinical outcomes were analyzed. For multivariate analysis, a binary logistic regression model was performed. Log-rank test was used to compare the cumulative incidence of hepatocellular carcinoma (HCC) in NAs-treated and non-NA streated patients. Results: Baseline liver histology status showed that 58.6% (58/99) patients had obvious liver tissue damage in their baseline liver tissue pathology (G≥2 and /or S≥2). Univariate logistic regression analysis showed that a liver cirrhosis (LC) family history, a HBsAg-positive family history, baseline alanine aminotransferase and aspartate aminotransferase levels were positively correlated factors for liver tissue damage. Multivariate logistic regression analysis showed that a LC family history was the main risk factor for liver tissue damage. Twenty-three cases had received a second liver biopsy after an interval of 4.5 years. In 10 untreated cases, the second liver biopsy results showed the rate of obvious liver tissue damage (G≥2 and/ or S≥2) increased from 50.0% to 90.0%. In the other 13 cases who received NAs treatment, the second liver biopsy showed improvement in liver histology, and the rate of obvious liver tissue damage decreased from 61.5% to 46.2%. The 5-year HCC cumulative incidence in non-NAs-treated patients was significantly higher than that of in NAs-treated patients (17.7% vs. 3.8%, P = 0.046). Conclusion: For most HBeAg-negative CHB patients with low viral load, liver tissue pathology result suggests that it meets the indications for antiviral therapy, especially in patients with a LC familial history. Without antiviral therapy, liver tissue damage for these patients will progressively worse with the high incidence of HCC. Therefore, it is suggested that antiviral therapy should be started as soon as possible for the HBeAg-negative CHB patients with low viral load regardless of the alanine aminotransferase level, especially in patients over 30 years-old with a LC or HCC family history.

[Mechanism of salicylic acid ameliorates salt-induced changes in Andrographis paniculata].

In this study, Andrographis paniculata seedlings were used as experimental materials to study the effects of salicylic acid(SA) on the growth and effective component accumulation of A. paniculata under NaCl stress. The results showed that with the increase of NaCl concentration, the growth of A. paniculata seedlings was significantly inhibited, and the content of carotene and carotenoid decreased. The activity of antioxidant enzyme was enhanced. At the same time, the contents of proline, proline and soluble protein were on the rise. The contents of andrographolide, new andrographolide and deoxyandrographolide showed an upward trend, while deoxyandrographolide showed a downward trend. Treatment with 100 mmol·L~(-1) NaCl+5 mg·L~(-1) SA showed a significant increase in antioxidant enzyme activity in A. paniculata leaves. Treatment with 100 mmol·L~(-1) NaCl+10 mg·L~(-1) SA showed significant changes in soluble protein and proline content in A. paniculata leaves, while MDA content in A. paniculata leaves significantly decreased. 10 mg·L~(-1) SA had the best effect on the growth of A. paniculata seedlings under salt stress. Under the treatment of 50 mmol·L~(-1) NaCl+10 mg·L~(-1) SA, fresh weight, dry weight and leaf dry weight of A. paniculata seedlings reached the highest level, which were 1.02, 1.09 and 1.11 times of those in the control group, respectively. The concentrations of NaCl and 10 mg·L~(-1) SA were significantly higher than those of the control group. Four key enzyme genes of A. paniculata diterpene lactone synthesis pathway were selected to explore the molecular mechanism of salicylic acid to alleviate salt stress. With the increase of salt stress, the relative expressions of HMGR, GGPS and ApCPS were up-regulated, indicating that salt stress may enhance the synthesis of A. paniculata diterpene lactone through MVA pathway. SA can effectively promote the growth and development of A. paniculata under salt stress, improve its osmotic regulation and antioxidant capacity, improve its salt tolerance, and alleviate the effects of salt stress on A. paniculata.

p38-Regulated/activated protein kinase plays a pivotal role in protecting heart against ischemia-reperfusion injury and preserving cardiac performance.

p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK-/- and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK-/- mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK-/- mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK-/- mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.

Irisin promotes cardiac progenitor cell-induced myocardial repair and functional improvement in infarcted heart.

Irisin, a newly identified hormone and cardiokine, is critical for modulating body metabolism. New evidence indicates that irisin protects the heart against myocardial ischemic injury. However, whether irisin enhances cardiac progenitor cell (CPC)-induced cardiac repair remains unknown. This study examines the effect of irisin on CPC-induced cardiac repair when these cells are introduced into the infarcted myocardium. Nkx2.5+ CPC stable cells were isolated from mouse embryonic stem cells. Nkx2.5 + CPCs (0.5 × 10 6 ) were reintroduced into the infarcted myocardium using PEGlylated fibrin delivery. The mouse myocardial infarction model was created by permanent ligation of the left anterior descending (LAD) artery. Nkx2.5 + CPCs were pretreated with irisin at a concentration of 5 ng/ml in vitro for 24 hr before transplantation. Myocardial functions were evaluated by echocardiographic measurement. Eight weeks after engraftment, Nkx2.5 + CPCs improved ventricular function as evident by an increase in ejection fraction and fractional shortening. These findings are concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Transplantation of Nkx2.5 + CPCs promoted cardiac regeneration and neovascularization, which were increased with the pretreatment of Nkx2.5 + CPCs with irisin. Furthermore, irisin treatment promoted myocyte proliferation as indicated by proliferative markers Ki67 and phosphorylated histone 3 and decreased apoptosis. Additionally, irisin resulted in a marked reduction of histone deacetylase 4 and increased p38 acetylation in cultured CPCs. These results indicate that irisin promoted Nkx2.5 + CPC-induced cardiac regeneration and functional improvement and that irisin serves as a novel therapeutic approach for stem cells in cardiac repair.

[Comparison of the minimally invasive treatments of laparoscopic and endosopic for common bile duct stones after gastrojejunostomy].

OBJECTIVE: To explore the feasibility and to compare the merits and demerits of laparoscopic and endoscopic approach in removing common bile duct stones in patients with gastrojejunostomy after gastrectomy. METHODS: Between January 2012 and December 2016, 25 patients with common bile duct stones after gastrojejunostomy received laparoscopic or endoscopic treatment in our centers. They were divided into laparoscopic group and endoscopic group based on treatment approaches for common bile duct stones, including 15 patients in laparoscopic group and 10 in endoscopic group. The clinical characteristics and outcomes between the two groups were retrospectively analyzed. RESULTS: Among the 25 patients with gastrojejunostomy, the method of reconstruction was Billroth II in 21 patients and Roux-en-Y in 4 patients. Six patients received laparoscopic or endoscopic treatment during the acute cholangitis state. Among the laparoscopic group, 5 patients with stones more than 1 cm, 7 patients with multiple stones, while in the endoscopic group, 3 patients with stones more than 1 cm and 4 patients with multiple stones. Fourteen patients in the laparoscopic group with coexisting gallbladder stones, and 6 of their common bile duct stones were successfully removed by transcystic approach without T tube drainage. Stone removals were successful in 4 patients of the endoscopic group by a single performance, including 3 patients with single small stone and one patient with multiple small stones. Two patients in the laparoscopic group were converted to open surgery for severe adhesion and one patient in the endoscopic group turned to laparoscopic operation for failing of finding papilla in the Roux-en-Y anastomotic status. The median hospital stays were 12 d and 10 d, respectively in the laparoscopic and endoscopic group. There were 3 patients with postoperative complications, including one patient with paralytic ileus in the laparoscopic group and 2 patients with biliary pancreatitis or bacteremia in the endoscopic group, and all of them recovered uneventfully with conservative treatment. CONCLUSION: Both laparoscopic and endoscopic approaches are feasible for removing stones in the common bile duct in patients with gastrojejunostomy after gastrectomy, and they complement each other. In addition, both techniques are difficult to conduct, and a technical competence should be considered in selection of each method.

[Laparoscopic treatment for incidental gallbladder cancer: a retrospective 10 years study from a single institution].

Objective: To explore the feasibility of laparoscopic treatment for incidental gallbladder cancer(IGBCA) and analyze the factors influencing prognosis. Methods: A retrospective study of 71 patients with IGBCA received laparoscopic treatment at Department of General Surgery, Peking University Third Hospital from January 2007 to December 2016 was conducted,the clinicopathological data and prognosis were analyzed. There were 18 males and 53 females,aged 23 to 81 years. They were divided into two groups based on the presence of intraluminal mass in the gallbladder. Sixty-five of the 71 patients received laparoscopic radical resection, the prognosis of them were compared with 14 patients with open radical resection. Results: Among the 71 patients,65 patients received radical resection,3 patients simple gallbaldder resection and 3 patients palliative resection. Postoperative complications occurred in 6 patients. IGBCA were detected by frozen section in 57 patients,with the accuracy of 96.5%,while the accuracy of T stage is 43.8% in the 48 patients received T stage evaluation during frozen section examination. The T stages based on final pathology were Tis(n=6),T1a(n=5),T1b(n=10),T2(n=46),and T3(n=4).The number of harvested lymph node was 4.7±2.9(range:2-12).There are 14 patients with lymph node metastasis. The 50 patients with intraluminal gallbladder mass include 21 patients with ≤T1b stage and 29 patients with ≥T2 stage, while the 21 patients without intraluminal gallbladder mass are all with ≥T2 stage. The median survival time of the 71 patients was 33 months, with the 5-year cumulative survival rate 67.3%. The 5-year cumulative survival rate is 78.5% for the 65 patients who received radical resection,comparable with those who received open radical resection(P=0.485).Univariate analysis demonstrated that T stage, lymph node metastasis, G grade, lymphovascular invasion, neural invasion, acute cholecystectomy, bile spillage, gallbladder mass and preoperative CA19-9/CEA were the most important prognostic factors(P<0.05). Conclusions: Laparoscopic treatment for IGBCA is feasible, especially for those with intraluminal gallbladder mass. The accuracy of frozen section examination in evaluating T stage is low.

cAMP attenuates TGF-ß's profibrotic responses in osteoarthritic synoviocytes: involvement of hyaluronan and PRG4.

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-ß to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4-/- mouse exhibits synovial hyperplasia. We investigated the antifibrotic effects of increased intracellular cAMP in TGF-ß-stimulated human OA synoviocytes. TGF-ß1 stimulated collagen I (COL1A1), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, and procollagen I, α-SMA, HA, and PRG4 production, migration, and proliferation of OA synoviocytes were measured. Treatment of OA synoviocytes with forskolin (10 µM) increased intracellular cAMP levels and reduced TGF-ß1-stimulated COL1A1, α-SMA, and TIMP-1 expression, with no change in PLOD2 expression. Forskolin also reduced TGF-ß1-stimulated procollagen I and α-SMA content as well as synoviocyte migration and proliferation. Forskolin (10 µM) increased HA secretion and PRG4 expression and production. A cell-permeant cAMP analog reduced COL1A1 and α-SMA expression and enhanced HA and PRG4 secretion by OA synoviocytes. HA and PRG4 reduced α-SMA expression and content, and PRG4 reduced COL1A1 expression and procollagen I content in OA synoviocytes. Prg4-/- synovium exhibited increased α-SMA, COL1A1, and TIMP-1 expression compared with Prg4+/+ synovium. Prg4-/- synoviocytes demonstrated strong α-SMA and collagen type I staining, whereas these were undetected in Prg4+/+ synoviocytes and were reduced with PRG4 treatment. We conclude that increasing intracellular cAMP levels in synoviocytes mitigates synovial fibrosis through enhanced production of HA and PRG4, possibly representing a novel approach for treatment of OA synovial fibrosis.

[Application of purse string suture with Harmonious Clips and Olympus endoloop in single channel endoscopy for large gastric antrum mucosa defect].

Objective: To evaluate the feasibility and safety of purse string suture with Harmonious Clips and Olympus endoloop in single channel endoscopy for large gastric antrum mucosa defect. Methods: A total of 33 patients who underwent ESD of gastric antrum in single channel endoscopy at the First people's Hospital of Wujiang District from January 2015 to April 2018 were retrospectively analyzed. Everyone had one lesion, and the diameters were all more than 3 cm. After the resection and hemostasis, purse string suture with Harmonious Clips and Olympus endoloop or no suture in study group (n=16) and the control group (n=17). The degree of abdominal pain, postoperative gastrointestinal decompression time, incidence of delayed hemorrhage, postoperative hospital stay and the healing rate were observed and compared. Results: All patients successfully completed resection, no perforation occurred, and all the lesions were resected completely in one time. All patients in the study group were sutured successfully. The abdominal pain score on the first day after operation was (2.7±0.7) in the study group, and (3.6±0.8) in the control group(t=3.686, P=0.001). The mean time of postoperative gastrointestinal decompression was (1.6±0.5) days in the study group, while (2.4±0.7) days in the control group(t=3.675, P=0.001). No delayed bleeding occurred in the study group, while 5 cases in the control group had delayed bleeding. The rate of delayed hemorrhage was 29.4% in control group, 4 cases successfully achieved hemostasis through endoscopy therapy, 1 case was given surgical intervention after ineffective endoscopic hemostasis(P=0.044). The average postoperative hospital stay were (6.2±1.1) days and (5.9±2.0) days respectively (t=0.423, P=0.675). Two months after the operation, the two groups of patients reviewed the gastroscopy, the results showed that, all wounds in the study group were healed completely, the healing rate was 100%. There were 6 cases of incomplete wound healing in the control group, the healing rate was 64.7%(P=0.018). No recurrence was found after 6 months of operation through reviewing gastroscopy. Conclution: It indicates good feasibility and efficacy about the purse string suture with Harmonious Clip and Olympus endoloop in single channel endoscopy for large gastric antrum mucosa defect. It is safe and reliable, worth of being generalized.

[Effect of Wnt/ß-catenin signal regulating EMT level on the differentiation of mouse ESC to liver tissue].

Objective: To study embryonic stem cell (ESC) differentiation into liver tissue structure from the perspective of epithelial mesenchymal transition (EMT). Methods: ESC of Balb/c mice was selected to induced into hepatic cell using hepatocyte growth factor (HGF), fibroblast growth factor (FGF) in vitro, and at the time points of metaphase (13 d) and maturity (17 d) of differentiation, dynamic inhibition of Wnt/ß-catenin signal was made to reduce the level of EMT. Finally, three-dimensional organization structure growth of the differentiation cells was observed in the differentiation system.Expressions of the liver cells vascular markers[albumin (Alb) and vascular endothelial growth factor (VEGFR)]were detected. Results: During the differentiation of ESC, the level of early EMT in the experimental group and the control group was not significantly different. The level of mid-late EMT in the experiment group was significantly lower than the control group. On the day 18 and 20 of differentiation, the relative mRNA expression level of E-cadherin was 0.61±0.15 and 0.47±0.05 in the experimental group, and 0.07±0.05 and 0 in the control group, respectively.The expression level of ALB/AFP/CK8/CK19 in the experimental group was generally higher than that of the control group in the same period, while CD31/VEGFR1 markers in the experimental group decreased more slowly in the late period of differentiation compared with the control group. In the supernatant of ESC culture, the Alb of the experimental group could be detected onday 7, and the concentration was (0.32±0.02) mg/L, while Alb in the control group was (0.19±0.05) mg/L. Urea in the experimental group could be detected on the day 13, and the concentration was (8.7 ±1.0) µmol/L, and the urea concentration of the control group was (3.1±1.2) µmol/L. The concentration of Alb and urea in the culture supernatant of ESC differentiation system increased significantly with the prolongation of the differentiation time, and the Alb and urea concentrations in the experimental group were significantly higher than those in the control group at the same time period. In addition, the differentiated cells in the experimental group could maintain the growth of three-dimensional tissue, while the differentiated cells in the control group eventually showed a single cell state. The expression of hepatic and vascular cell markers could be detected in the experimental group. Immunofluorescence results showed that the hepatocytes and vascular structures were tightly arranged. HE staining showed the formation of hepatic lobular structure, while the control group had no vascular component markers. Conclusion: The differentiation of ESC into liver tissue can be effectively promoted by decreasing the level of EMT at the mid-late stage of ESC differentiation.