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.

2022 Chinese national clinical practice guideline on Helicobacter pylori eradication treatment.

BACKGROUND: Helicobacter pylori ( H. pylori ) infection is an infectious disease with a prevalence rate of up to 50% worldwide. It can cause indigestion, gastritis, peptic ulcer, and gastric cancer. H. pylori eradication treatment can effectively control disease progression and reduce the risk of the above conditions. However, the escalating trend of antibiotic resistance presents a global challenge for H. pylori eradication. We aim to provide guidance on pharmacological treatment of H. pylori infection. METHODS: This clinical practice guideline is developed following the World Health Organization's recommended process, adopting Grading of Recommendations Assessment, Development and Evaluation in assessing evidence quality, and utilizing Evidence to Decision framework to formulate clinical recommendations, minimizing bias and increasing transparency of the clinical practice guideline development process. We used the Reporting Items for practice Guidelines in HealThcare (RIGHT) statement and The Appraisal of Guidelines for Research and Evaluation II (AGREE II) as reporting and conduct guides to ensure the guideline's completeness and transparency. RESULTS: Though decreasing in developed countries, the prevalence of H. pylori remains high in developing countries, causing a major public health burden. This clinical practice guideline contains 12 recommendations concerning pharmacological treatment for H. pylori eradication. Among them, it is worth highlighting that bismuth preparations are inexpensive, safe, and effective, consequently making bismuth quadruple therapy a preferred choice for initial and rescue treatment. In empirical treatment, high-dose dual therapy is equally effective compared with bismuth quadruple therapy. CONCLUSIONS: The 12 recommendations in this clinical practice guideline are formed with consideration for stakeholders' values and preferences, resource use, feasibility, and acceptability. Recommendations are generalizable to resource limited settings with similar antibiotic resistance pattern as China, and lower middle-income countries facing comparable sociological and technical challenges. REGISTRATION: Guidelines International Network (GIN) website, https://guidelines.ebmportal.com/node/69996 .

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.

First evidence of pyrrolizidine alkaloid N-oxide-induced hepatic sinusoidal obstruction syndrome in humans.

Pyrrolizidine alkaloids (PAs) are among the most potent phytotoxins widely distributed in plant species around the world. PA is one of the major causes responsible for the development of hepatic sinusoidal obstruction syndrome (HSOS) and exerts hepatotoxicity via metabolic activation to form the reactive metabolites, which bind with cellular proteins to generate pyrrole-protein adducts, leading to hepatotoxicity. PA N-oxides coexist with their corresponding PAs in plants with varied quantities, sometimes even higher than that of PAs, but the toxicity of PA N-oxides remains unclear. The current study unequivocally identified PA N-oxides as the sole or predominant form of PAs in 18 Gynura segetum herbal samples ingested by patients with liver damage. For the first time, PA N-oxides were recorded to induce HSOS in human. PA N-oxide-induced hepatotoxicity was further confirmed on mice orally dosed of herbal extract containing 170 µmol PA N-oxides/kg/day, with its hepatotoxicity similar to but potency much lower than the corresponding PAs. Furthermore, toxicokinetic study after a single oral dose of senecionine N-oxide (55 µmol/kg) on rats revealed the toxic mechanism that PA N-oxides induced hepatotoxicity via their biotransformation to the corresponding PAs followed by the metabolic activation to form pyrrole-protein adducts. The remarkable differences in toxicokinetic profiles of PAs and PA N-oxides were found and attributed to their significantly different hepatotoxic potency. The findings of PA N-oxide-induced hepatotoxicity in humans and rodents suggested that the contents of both PAs and PA N-oxides present in herbs and foods should be regulated and controlled in use.

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.

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.

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.

[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.

Fast determination of paeonol in plasma by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry.

Paeonol is the active component in the traditional Chinese medicines (TCMs), such as Cynanchum paniculatum, which has been used to treat many diseases, such as eczema. In this work, a simple, rapid and sensitive method was developed for the determination of paeonol in rabbit plasma, which was based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). The extraction parameters of fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized; the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 microg mL(-1), R(2)>0.990), low detection limit (2.0 ng mL(-1)) and good precision (R.S.D. value less than 8%). Finally, GC/MS following HS-SPME was applied to fast determination of paeonol in rabbit plasma at different time point after oral demonstration of Cynanchum paniculatum essential oil. The experimental results suggest that the proposed method provided an alternative and novel approach to the pharmacokinetics study of paeonol in the TCMs.

Gas chromatography-mass spectrometry following pressurized hot water extraction and solid-phase microextraction for quantification of eucalyptol, camphor, and borneol in Chrysanthemum flowers.

Chrysanthemum flower is a common traditional Chinese medicine (TCM). In this work, pressurized hot water extraction (PHWE) followed by headspace solid-phase microextraction (HS-SPME) and GC-MS was developed for the determination of three main active volatile compounds of eucalyptol, camphor, and borneol in Chrysanthemum flowers from four different growing areas in China by internal standard method. The parameters of PHWE and HS-SPME were optimized. The method was also validated. The results showed that PHWE-SPME-GC-MS is a simple, rapid, efficient, and solvent-free technique for the quantitative determination of eucalyptol, camphor, and borneol in TCMs and is potentially useful for the TCM quality assessment.

The origin of C1A-C2 interdomain interactions in protein kinase Calpha.

The regulatory domain of protein kinase Calpha (PKCalpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCalpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCalpha revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCalpha, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCalpha C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCalpha activation.

[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.