The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo.

OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China.

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.

Phosphocreatine attenuates Gynura segetum-induced hepatocyte apoptosis via a SIRT3-SOD2-mitochondrial reactive oxygen species pathway.

Purpose: To investigate the mitochondria-related mechanism of Gynura segetum (GS)-induced apoptosis and the protective effect of phosphocreatine (PCr), a mitochondrial respiration regulator. Methods: First, the mechanism was explored in human hepatocyte cell line. The mitochondrial oxidative stress was determined by fluorescence assay. The level of sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), SOD2, and apoptosis were detected by Western blotting. Mito-TEMPO and cell lines of viral vector-mediated overexpression of SIRT3 and SIRT3H248Y were used to further verify the mechanism of GS-induced apoptosis. GS-induced liver injury mice models were built by GS through intragastric administration and interfered by PCr through intraperitoneal injection. A total of 30 C57BL/6J mice were assigned to 5 groups and treated with either saline, PCr (100 mg/kg), GS (30 g/kg), or PCr (50 or 100 mg/kg)+GS (30 g/kg). Liver hematoxylin and eosin (HE) staining, immunohistochemical analysis, and blood biochemical evaluation were performed. Results: GS induced hepatocyte apoptosis and elevated levels of mitochondrial ROS in L-02 cells. The expression of SIRT3 was decreased. Downregulation of SIRT3 was associated with increased levels of Ac-SOD2, which is the inactivated enzymatic form of SOD2. Conversely, when overexpressing SIRT3 in GS-treated cells, SOD2 activity was restored, and mitochondrial ROS levels and hepatocyte apoptosis declined. Upon administration of PCr to GS-treated cells, they exhibited a significant upregulation of SIRT3 and were protected against apoptosis. In animal experiments, serum ALT level and mitochondrial ROS of the mice treated with GS and 50 mg/kg PCr were significantly attenuated compared with only GS treated. The changes in SIRT3 expression were also consistent with the in vitro results. In addition, immunohistochemical analysis of the mouse liver showed that Ac-SOD2 was decreased in the PCr and GS co-treated group compared with GS treated group. Conclusion: GS caused liver injury by dysregulating mitochondrial ROS generation via a SIRT3-SOD2 pathway. PCr is a potential agent to treat GS-induced liver injury by mitochondrial protection.

CSH guidelines for the diagnosis and treatment of drug-induced liver injury.

Drug-induced liver injury (DILI) is an important clinical problem, which has received more attention in recent decades. It can be induced by small chemical molecules, biological agents, traditional Chinese medicines (TCM), natural medicines (NM), health products (HP), and dietary supplements (DS). Idiosyncratic DILI is far more common than intrinsic DILI clinically and can be classified into hepatocellular injury, cholestatic injury, hepatocellular-cholestatic mixed injury, and vascular injury based on the types of injured target cells. The CSH guidelines summarized the epidemiology, pathogenesis, pathology, and clinical manifestation and gives 16 evidence-based recommendations on diagnosis, differential diagnosis, treatment, and prevention of DILI.

Comparative effectiveness and tolerance of treatments for Helicobacter pylori: systematic review and network meta-analysis.

OBJECTIVE: To determine the most efficacious treatment for eradication of Helicobacter pylori with the lowest likelihood of some common adverse events among pre-recommended and newer treatment regimens. DESIGN: Systematic review and network meta-analysis. DATA SOURCES: Cochrane Library, PubMed, and Embase without language or date restrictions. STUDY SELECTION: Full text reports of randomised controlled trials that compared different eradication treatments for H pylori among adults. RESULTS: Of the 15,565 studies identified, 143 were eligible and included. Data on 14 kinds of treatments were available. Of 91 possible comparisons for the efficacy outcome, 34 were compared directly and the following treatments performed better: seven days of concomitant treatment (proton pump inhibitor and three kinds of antibiotics administered together), 10 or 14 days of concomitant treatment, 10 or 14 days of probiotic supplemented triple treatment (standard triple treatment which is probiotic supplemented), 10 or 14 days of levofloxacin based triple treatment (proton pump inhibitor, levofloxacin, and antibiotic administered together), 14 days of hybrid treatment (proton pump inhibitor and amoxicillin used for seven days, followed by a proton pump inhibitor, amoxicillin, clarithromycin, and 5-nitroimidazole for another seven days), and 10 or 14 days of sequential treatment (five or seven days of a proton pump inhibitor plus amoxicillin, followed by five or seven additional days of a proton pump inhibitor plus clarithromycin and 5-nitroimidazole or amoxicillin). In terms of tolerance, all treatments were considered tolerable, but seven days of probiotic supplemented triple treatment and seven days of levofloxacin based triple treatment ranked best in terms of the proportion of adverse events reported. CONCLUSION: Comparison of different eradication treatments for H pylori showed that concomitant treatments, 10 or 14 days of probiotic supplemented triple treatment, 10 or 14 days of levofloxacin based triple treatment, 14 days of hybrid treatment, and 10 or 14 days of sequential treatment might be better alternatives for the eradication of H pylori.

Tusanqi and hepatic sinusoidal obstruction syndrome.

Hepatic sinusoidal obstruction syndrome (HSOS), characterized by hepatomegaly, ascites and hyperbilirubinemia, is caused by toxic injury to hepatic sinusoidal endothelial cells. One major etiology of HSOS in China is the intake of products containing pyrrolizidine alkaloids (PA) such as Tusanqi. The manifestations of patients with HSOS are usually non-specific, presenting with abnormal liver function and portal hypertension. Diagnosis of the disease depends mostly on liver histopathology when clinical and imaging data are not sufficient. A history of Tusanqi intake is mostly important for the diagnosis. Due to a lack of effective, evidence-based treatments for HSOS, avoiding the mistaken use of PA-containing products including Tusanqi is important for the prevention of HSOS.

Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats.

BACKGROUND: At present there is no effective and accepted therapy for hepatic fibrosis. Transforming growth factor (TGF)-ß1 signaling pathway contributes greatly to hepatic fibrosis. Reducing TGF-ß synthesis or inhibiting components of its complex signaling pathway represent important therapeutic targets. The aim of the study was to investigate the effect of curcumin on liver fibrosis and whether curcumin attenuates the TGF-ß1 signaling pathway. METHODS: Sprague-Dawley rat was induced liver fibrosis by carbon tetrachloride (CCl4) for six weeks together with or without curcumin, and hepatic histopathology and collagen content were employed to quantify liver necro-inflammation and fibrosis. Moreover, the mRNA and protein expression levels of TGF-ß1, Smad2, phosphorylated Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) were determined by quantitative real time-PCR, Western blot, or immunohistochemistry. RESULTS: Rats treated with curcumin improved liver necro-inflammation, and reduced liver fibrosis in association with decreased α-smooth muscle actin expression, and decreased collagen deposition. Furthermore, curcumin significantly attenuated expressions of TGFß1, Smad2, phosphorylated Smad2, Smad3, and CTGF and induced expression of the Smad7. CONCLUSIONS: Curcumin significantly attenuated the severity of CCl4-induced liver inflammation and fibrosis through inhibition of TGF-ß1/Smad signalling pathway and CTGF expression. These data suggest that curcumin might be an effective antifibrotic drug in the prevention of liver disease progression.

Protective effects of curcumin against hepatic fibrosis induced by carbon tetrachloride: modulation of high-mobility group box 1, Toll-like receptor 4 and 2 expression.

The aim of the study was to investigate the effect of curcumin on the liver fibrosis induced by carbon tetrachloride (CCl(4)) in rats, and to elucidate its underlying molecular mechanisms. Rats were administered with CCl(4) together with or without curcumin for 6 weeks. Hepatic damage was evaluated by analysis of liver function tests in serum. Hepatic histopathology and collagen content were employed to quantify liver fibrosis; and activated hepatic stellate cells were assessed. Moreover, the mRNA and protein expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, high-mobility group box 1 (HMGB1), Toll like receptor (TLR) 2 and TLR4 were determined by quantitative real time PCR, Western blot or immunohistochemistry. Treatment with curcumin significantly attenuated CCl(4)-induce liver injury, hepatic inflammation and reduced the levels of proinflammatory mediators (TNF-α, IL-6 and MCP-1). Moreover, curcumin significantly inhibited extracellular matrix deposition, reduced the number of activated stellate cells, and decreased the levels of HMGB1, TLR4 and TLR2 expression in the rat model of fibrogenesis. These results suggest that curcumin could be an effective agent for preventing liver fibrosis and its mechanism may in part be a consequence of the reduction TLR2, TLR4 and HMGB1 expression.

Definitive diagnosis of hepatic sinusoidal obstruction syndrome induced by pyrrolizidine alkaloids.

OBJECTIVE: Hepatic sinusoidal obstruction syndrome (HSOS) induced by a Chinese medicinal herb Tusanqi is increasingly being reported in recent years. The aim of the study was to investigate the possibility of using blood pyrrole-protein adducts test as a confirmatory diagnostic method. METHODS: Patients with HSOS according to international diagnostic criteria associated with Tusanqi from January 2006 to August 2010 in Zhongshan Hospital Fudan University were included and clinical features were collected. Pyrrole-protein adducts in blood sample were determined with ultra performance liquid chromatography-mass spectrometry (UPLC-MS) while pyrrolizidine alkaloids (PAs) in available herbal preparations were analyzed by high performance liquid chromatography-ultraviolet (HPLC-UV). RESULTS: Five patients (age 41-72 years, median age 54 years, all women) were included. Ascites (5/5), jaundice (5/5) and hepatomegaly (4/5) were common manifestations. The imaging features were diffused, patchy hepatic enhancement, periportal edema and ascites. Pathology ascertained that blood flow was obstructive at the site of sinusoid. PAs (Seneionine and seneciphylline) were identified in all the three available herbal preparations ingested by the HSOS patients. Pyrrole-protein adducts were unequivocally found in all the five blood samples. Two patients recovered, two developed chronic illness and one died due to liver failure and hepatic encephalopathy. CONCLUSIONS: The detection of blood pyrrole-protein adducts using a UPLC-MS approach is a specific, reliable, unambiguous and confirmatory test for HSOS induced by PA, and should be used together with the conventional HSOS clinical diagnostic criteria for the definitive diagnosis of PA-induced HSOS.

Hepatic sinusoidal obstruction syndrome associated with consumption of Gynura segetum.

BACKGROUND & AIMS: One major cause of hepatic sinusoidal obstruction syndrome (HSOS) is the intake of pyrrolizidine alkaloid (PA)-containing products. Over 8000 PA-induced HSOS cases have been reported worldwide and at least 51 among them were suspected to be attributed to exposure to the Chinese medicine 'Tusanqi'. PA-induced hepatotoxicity involves cytochrome P450-mediated metabolic activation of PAs to electrophilic pyrrolic metabolites which react with macromolecules, such as proteins. However, no studies have found such protein adduction in HSOS patients. We report one HSOS case confirmed by liver biopsy, where the patient claimed taking 'Tusanqi' as self-medication. METHODS: The herb was analyzed by HPLC-MS, and its induced hepatotoxicity in rats was assessed by monitoring the alteration of serum ALT level and liver morphology. Blood pyrrole-protein adducts were determined by UPLC-MS. RESULTS: The herb the patient consumed was identified as Gynura segetum, an erroneous substitute of non-PA-containing Sedum aizoon, called 'Tusanqi'. Hepatotoxic PAs senecionine and seneciphylline were detected in G. segetum. In the PA-exposed patient, serum pyrrole-protein adducts were detected by a newly developed analytical approach. The animal study showed a good correlation of liver injury with the ingestion of G. segetum. CONCLUSIONS: For the first time, serum pyrrole-protein adducts were unequivocally detected in a PA-induced HSOS patient, and such adducts show a potential to be developed as a biomarker for the assessment of PA-induced HSOS. Similar to the well-known case of aristolochic acid-poisoning, the observed HSOS was confirmed to arise from the consumption of PA-containing G. segetum, an erroneous substitute of non-PA-containing S. aizoon.

Early diagnosis and therapeutic choice of Klebsiella pneumoniae liver abscess.

Nowadays, pyogenic liver abscess (PLA) is still a common and severe intra-abdominal infection, and Klebsiella pneumoniae had emerged as the most common pathogenic bacteria worldwide in the past ten years. Our study aims to achieve an early pathogenic diagnosis and rational therapy modality for Klebsiella pneumoniae liver abscess (KLA) through clinical data analysis. A total of 197 inpatients in Zhongshan Hospital, Shanghai, diagnosed as having liver abscess between March 2001 and September 2009 were enrolled. Patients with monomicrobial infection were divided into two groups: patients with K. pneumoniae liver abscess (KLA group, n=106) and those with non-Klebsiella pneumoniae liver abscess (NKLA group, n=56). A retrospective analysis was made between these two groups on the aspects of underlying diseases, clinical characteristics, laboratory data, culture results, and imaging findings. To evaluate the effects of different medical interventions, monomicrobial KLA patients were further divided into four subgroups (percutaneous liver aspiration, aspiration plus antibiotics flushing, aspiration plus retained catheter, and aspiration plus antibiotics flushing and retained catheter), and corresponding therapeutic effects were analyzed. KLA was more likely to occur in patients with coexisting diseases such as diabetes mellitus (53.77% vs 25.00%, P=0.001) and hepatic adipose infiltration (16.04% vs 5.36%, P=0.029). Compared to NKLA group, clinical characteristics including abdominal pain (40.57% vs 57.14%, P=0.044), hypodynamia (19.81% vs 46.43%, P=0.001), and hepatomegaly (4.72% vs 14.29%, P=0.033) were much milder, but with a higher fasting blood glucose level (7.84±0.36 vs 5.76±0.30, P=0.001) on admission in KLA group. In addition, KLA abscess often appeared singly in the right lobe of the liver with gas forming nature (32.88% vs 13.51%, P=0.039), unsmooth rim (71.23% vs 40.54%, P=0.002), and dynamic septum enhancement (41.10% vs 16.22%, P=0.009). Compared to mono aspiration subgroup, additional antibiotic flushing could not further improve clinical outcomes of KLA patients (P>0.05); however, the retained catheter showed obvious advantage in reducing abscess diameter (34.38±3.25 mm vs 22.67±2.37 mm, P=0.017). It can be concluded that the strong association with diabetes, milder clinical symptoms, and gas-forming nature in CT images makes early pathogenic diagnosis of KLA possible. Comparatively, ultrasonography-guided percutaneous liver aspiration with retained catheter may be the most rational intervention modality of KLA.

The efficacy of Combizym in the treatment of Chinese patients with dyspepsia: a multicenter, randomized, placebo-controlled and cross-over study: Shanghai Combizym Clinical Cooperative Group.

OBJECTIVE: To evaluate the efficacy and safety of Combizym treatment in Chinese patients with dyspepsia. METHODS: In this multicenter, randomized, placebo-controlled cross-over study, a total of 151 patients (76 men and 75 women, mean age: 44.67 +/- 6.46 years, range: 22-67 years) with dyspepsia whose symptoms were not relieved by placebos were recruited. They were randomly divided into group A (79 patients, 2 weeks of Combizym treatment, two tablets post-meal, t.i.d.; then 1 week of wash-out, followed by 2 weeks of placebo treatment, two tablets post-meal, t.i.d.) or group B (72 patients, 2 weeks of placebo treatment, two tablets post-meal, t.i.d. then one week of wash-out, followed by 2 weeks of Combizym treatment, 2 tablets post-meal, t.i.d.). The index of severity of the dyspepsia symptoms was evaluated before and after each treatment phase with Combizym or the placebo. RESULTS: Compared with the placebo, 2 weeks of Combizym treatment decreased the severity index of dyspepsia symptoms significantly (27.64 +/- 1.77 to 9.72 +/- 1.33 vs 23.99 +/- 1.28 to 22.03 +/- 1.40, P < 0.01). The efficacy rates of Combizym and the placebo on dyspepsia were 89.63% and 21.68%, respectively (P < 0.01). According to the improvement of symptoms index, individual dyspepsia symptoms that could be attenuated by Combizym therapy were anepithymia, abdominal distension, belching, diarrhea, abdominal pain, epigastric burning. None of patients reported adverse events during the study. CONCLUSION: Combizym treatment effectively ameliorates dyspepsia symptoms in Chinese patients, with satisfactory safety and compliance.

[Effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis: preliminary experimental study with rats].

OBJECTIVE: To study the effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis. METHODS: Twenty-six male S-D rats were randomly divided into three groups, cirrhotic model group (n = 10), treated with CCl4 (CCl4/olive oil: v/v = 1:1), Fuzheng Huayu Decoction intervention group (n = 10), treated with CCl4 + Fuzheng Huayu Decoction, and normal control group (n = 6), treated with olive oil only. After 8 weeks, blood sample was collected from the vena cava inferior to undergo bi-dimensional electrophoresis (2DE) and analysis by PDQuest 7.3 software. Differential protein spots were cut, enzyme hydrolysis was conducted, and peptide fragments extracted from the mixture underwent mass spectrometry with MALDI-TOF-TOF-MS. The liver fibrogenesis was assessed by digital image analysis instrument of Masson's trichrome stained sections. RESULTS: The fibrosis area of the Fuzheng Huayu Decoction was 9% +/- 4%, significantly smaller than that of the cirrhotic model group (12% +/- 5%, P < 0.05). Ten markedly changed protein spots were identified by MALDI-TOF-TOF-MS. Eight of the 10 proteins, including plasma glutathione peroxidase, plasma glutathione peroxidase precursor, prealbumin, haptoglobin, apolipoprotein A-IV precursor, complement C4, inter-alpha-inhibitor H4 heavy chain, and serine/threonine-protein kinase MARK1 (microtubule- affinity regulating kinase 1) were expressed very lowly in the cirrhotic model group while were expressed highly in the Fuzheng Huayu Decoction group. The expression of liver regeneration-related protein LRRG03 and vimentin increased in the cirrhotic model group, and reduced in the Fuzheng Huayu Decoction group. CONCLUSION: Some proteins related to oxidative stress, cell proliferation and transformation have changed in the plasma of cirrhosis induced by CCl4. Fuzheng Huayu Decoction promotes protein synthesis and plays an anti-fibrotic role by antioxidation and accommodation of cell proliferation and transformation.

[Cost-effective analysis of preventive treatment on diabetes].

OBJECTIVE: To evaluate the cost-effectiveness of preventive treatment on diabetes, using metformin or acarbose among patients with impaired glucose tolerance. METHODS: Using data from diabetes prevention program (DPP) and STOP-NIDDM study, we evaluated the cost of preventing one new onset of diabetes in Shanghai, and to compare its cost with the current treatment cost. RESULTS: If metformin was used for preventive treatment as in DPP study, a total cost of 69 122.95RMB was needed for preventing one new onset of diabetes in three years period. If acarbose was used for preventive treatment as in STOP-NIDDM, then 154 116.05RMB was the cost to prevent one diabetes in 3.3 years of treatment. However, if the generic metformin was used, the total cost was only 21 666.63RMB for the 3-years treatment. Data showed that the average cost for treating diabetes per year was 9143.70RMB in Shanghai. CONCLUSION: The total cost of diabetes prevention was formidable, although generic metformin showed the trend of cost-effective. The cost of drugs took the biggest part of the total cost. To choose the cheap but effective drug for treatment might save a large part of the cost. Further clinical research concerning the prevention of complications might provide us with more information on the cost-effectiveness of preventive treatment on diabetes.