Progresses and analysis in fibrinolytic drugs induced hemorrhage / 药学学报

At present, most clinical thrombolytic drugs are plasminogen activators, which are highly dependent on the plasminogen level of the patient. Therefore, the efficacy of those drugs is restricted. Unlike the conventional thrombolytic plasminogen activator drugs, fibrinolytic drugs have direct fibrinolytic activity. Thus, fibrinolytic drugs can directly dissolve the thrombus, and its thromlysis efficacy is not restricted by the patients' plasminogen. This is a new type of thrombolytic drug with higher thrombolytic efficiency and safety, and has become one of the research hotspots at present. Although more and more agents that can be used as fibrinolytic drugs have been discovered, only a few of them can successfully be applied in clinical practice. The mainly underlying reason is the risk of bleeding. In this paper, based on the latest research progress of fibrinolytic drugs, the bleeding mechanisms and coping strategies of fibrinolytic drugs were systematically reviewed, five types of bleeding mechanisms of fibrinolytic drugs were summarized, and three types of coping strategies were proposed. We hope our work can provide theoretical basis for the development of safer and more efficient fibrinolytic drugs.

Progress of the development of anti-COVID-19 drugs / 药学学报

Since the outbreak of the novel coronavirus (SARS-CoV-2) disease COVID-19 (also known as 2019-nCoV) caused by SARS-CoV-2 in the end of 2019, it has spread rapidly in worldwide. Besides developing effective vaccines, it is urgent to develop safe and effective anti-SARS-CoV-2 drugs to fight this disease. Paxlovid, molnupiravir, sotrovimab and bebtelovimab are urgently authorized by FDA have been proved to be effective against Omicron. This manuscript mainly reviews the recent progress of effective inhibitors against the virus in the world, including receptor inhibitors, antibodies, natural product inhibitors, synthetic inhibitors and broad-spectrum antiviral drugs that are effective against other RNA viruses.

Research advances on hypolipidemic effect of polysaccharides / 中国中药杂志

Hyperlipidemia is a systemic chronic metabolic disease caused by dyslipidemia in the body. It is an important risk factor of accelerating atherosclerosis, which will cause coronary heart disease, thrombus and other cardiovascular diseases, so it is a "invisible killer" for human health. Controlling and lowering blood lipids can reduce the risk of cardiovascular and cerebrovascular diseases. The current therapies for hyperlipidemia mainly include chemical synthetic medicines. However, long-term use of hypolipidemic drugs would cause various side effects, and the demand of effective and nontoxic drugs for hyperlipidemia patients is eager. Polysaccharide has attracted worldwide concerns due to its characteristics of good biocompatibility and less side effects. Polysaccharide is a kind of biological macromolecule which is widely found in plant cell walls, animal cell membranes and microorganism cell walls. A number of studies have shown that polysaccharides from natural materials have broad biological activities, such as anti-tumor, immunomodulatory, antioxidant, hypoglycemic, and hypolipidemic effects, with broad application prospect. This paper has reviewed and summarized the polysaccharides with hypolipidemic effect and their mechanisms which have been reported at home and abroad, hoping to provide certain reference for their development and application in lowering blood lipids.

The strategies of targeting therapy of hepatocellular carcinoma by adeno-associated virus / 药学学报

Hepatocellular carcinoma (HCC) is the most common liver cancer, which is also the second leading cause of death in cancer. With the development of molecular biology and technology, gene therapy has become a new potential method to treat the cancer. As a viral gene-delivery system, the adeno-associated virus (AAV) is the most promising delivery vehicle for its high efficiency of infection, low pathogenicity and low immunogenicity. However, AAV has a wide range of host that may lead to side effects. Targeted gene therapy can achieve site-specific and high efficient gene expression, which avoids toxicity of systemic and non-targeted gene expression to improve the safety and efficacy of gene therapy. In this review, we provide an overview of the pathogenesis of HCC and the characteristics of AAV. Moreover, we discuss the targeting strategies currently employed in the gene therapy for HCC with a focus on targeting the transductional, transcriptional and posttranscriptional levels. New strategies are proposed for improving the quality of life and survival rate of patients with HCC.

Relationship between hepatitis B virus X protein and hepatic cellular cancer / 药学学报

Hepatic cellular cancer (HCC) is one of the most common cancers in the world, which is a serious threat to human health and life quality. More than 700 000 people die of HCC each year on average, and its incidence increases in many countries. Chronic hepatitis B virus (HBV) infection has been identified as a dominant risk factor for HCC. The pathogenesis of HBV-induced hepatocarcinogenesis is, however, incom-pletely understood. Evidence currently available supports a key role of the HBV X protein (HBx) in the cancer transformation and malignant tumor metastasis. HBx is a multifunctional regulator that may cooperate with the host factors to exert its effects on transcription, signal transduction, cell cycle progression, apoptosis, protein degradation, expression of oncogene and anti-oncogene. This review presents the current knowledge in the molecular pathogenesis of HBx in the induction of HCC.

Expression, purification, and characterization of fusion protein TAT-cytoglobin / 生物工程学报

he aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30 ± 0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR = 98%), which was significantly different from Cygb treatment group (RGR = 79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.

Adeno-associated vector mediated intracellular biological activity of human Kallistatin / 药学学报

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.

Interaction of E3 ligase HUWE1 and eukaryotic translation initiation factor eIF4E / 药学学报

To explore the regulation of eIF4E, we screened the protein interacting with eIF4E from human cDNA library by using yeast two-hybrid system. Several clones interacting with eIF4E were identified. One of them was homologous with HUWE1 (HECT, UBA and WWE domain containing 1, also named as ARF-BP1, HECTH9 or HUWE1). Cell co-immunoprecipitation showed that eIF4E could bind to HUWE1 in mammalian cells. We also found that HUWE1 bearing the HECT domain is necessary for its association with eIF4E.

Strategy for keeping efficient expression and pharmacodynamics in reducing rAAV gene medicine immune response based on the decrease of vector dosage / 药学学报

How to reduce immune response is an unprecedented challenge for rAAV gene medicine. Recent studies suggested that lowering dosage of the vector used could reduce immune response caused by rAAV gene medicine. Nevertheless, it would also decrease the transgene expression, leading to failure of gene treatment. It is therefore important to take appropriate steps to maintain high gene expression level and pharmacodynamic, while the dosage of rAAV used is reduced. Here, steps to enhancing gene therapy, such as optimization of the administration, reconstruction of the viral vector and selection of the promoter, are discussed in order to achieve maximum outcome.

Ubiquitination of recombinant adeno-associated viral vector and its application / 浙江大学学报·医学版

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

Combinatorial RNAi and its application in cancer gene therapy / 药学学报

RNA interference (RNAi) has been proved as a novel approach for gene therapy. However, RNAi mono-therapy only aims at single gene, it therefore may ultimately fail to cure cancers caused by polygene variation. To overcome the deficiency of RNAi mono-therapy, "combinatorial RNA interference" (coRNAi) was put forward as a new strategy. By co-expressing the inducers of RNAi triggering single or multiple targets directly and other RNA- or protein-based silencers, coRNAi keeps target genes silent, prevents carcinogenic progression and induces apoptosis of tumor cells. This paper mainly reviews the major strategies of coRNAi and their applications in cancer gene therapy.

Research progresses of plant polysaccharides on cell adhesion / 药学学报

Cell adhesion mediated by cell adhesion molecules (CAMs) constitutes essential life phenomenon. In inflammation, immunity, infection, thrombosis, tumor metastasis and wound healing, cell adhesion comes into being the basic physiological and pathological process. Intervening with cell adhesion has been the important therapeutic and prophylactic strategies for diseases. Accumulated evidence has indicated that plant polysaccharides especially those exacted from Chinese traditional and herbal drugs displayed various pharmacological effects such as anti-inflammation, anti-cancer, anti-infection, immunomodulation, cardiovascular protective effects and so on. In this paper, the research progress of plant polysaccharides on cell adhesion is reviewed.

Engineering and screening of artificial riboswitch as a novel gene control element / 生物工程学报

Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.

Recombinant adeno-associated virus vector related impurities / 生物工程学报

Recombinant adeno-associated virus (rAAV)-based vectors that can stably express therapeutic genes in vivo without detectable side-effect have shown great promise for human gene therapy. A major challenge for translation of promising research to clinical development is how to establish clinically compatible purification methods in separating rAAV from potentially pathogenic impurities, especially rAAV vector-related impurities, a class of impurities corresponding to AAV particles that closely resemble bona fide vectors and are difficult to remove. In this review we summarize the assembly process of rAAV vector-related impurities and their characteristics differed with rAAV vectors, and evaluate several current technologies to prevent their formation or separate them from rAAV stocks.

Capsid assembly and DNA encapsidation of adeno-associated virus / 生物工程学报

Recombinant adeno-associated viral vectors (rAAV) have been widely used as gene therapy vectors in clinical trials. Here, we reviewed the genomic structures and replication mechanisms of wt-AAV. Then, the assembly of capsid and the encapsidation of genomic DNA, two major events during AAV pakaging, was discussed in detail. Although the overall pattern of virus assembly and encapsidation is known, the molecular mechanisms and the structure-function relationship involved in these processes are not well understood. Further elucidatation of these processes may improve the production technology of rAAV and develop gene drug based on rAAV.

Role of cytoglobin in protecting hepatic stellate cells against oxidation induced damage / 生物工程学报

The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.

Selection and anti-cancer effects of siRNAs targeting HMGA2 gene / 药学学报

High mobility group A2 protein (HMGA2), an architectural factor, is highly expressed in various cancer types including lung cancers. It is a candidate target for cancer therapy. RNAi is an effective gene silencing method with low cost and less time-consuming. It is possible to exploit this technology in therapy. Here, 5 siRNAs targeting Hmga2 gene (HMGA2 siRNA1-5) were designed and synthesized. MTT assay, colony formation assay, transwell assay and flow cytometry were used to evaluate the effects of these siRNAs on lung cancer cell lines (NCI-H446 and A549). Results from cell proliferation, clone formation, migration and apoptosis showed that HMGA2 siRNA1, 3, 5 could affect these aspects for both lung cancer cell lines. Among the five siRNAs, HMGA2 siRNA5 showed the greatest inhibition effects. The inhibition effects of HMGA2 siRNA5 are sequence specific and are not due to the induction of interferon response. Taken together, siRNAs targeting Hmga2 gene are potential candidates for lung cancer gene therapy.

The asymmetric division and tumorigenesis of stem cells / 癌症

Stem cells use asymmetric and symmetric cell division to generate progeny. Symmetric cell division is defined as the generation of daughter cells that are destined to acquire the same fate. Stem cells divide asymmetrically to generate one daughter with a stem-cell fate and one daughter with different fate. Disruption of the machinery that regulates asymmetric division may be a reason for the generation of cancer. The asymmetric mechanism is maintained by cell polarity factors, cell fate determinants, and the spindle apparatus. The mutation or dysregulation of these factors may change stem cells from asymmetric to symmetric cell division, then leading to tumorigenesis. Therefore, further study is needed on the mechanisms of stem cell control between asymmetric and symmetric cell division, as well as the relationships among stem cells, cancer stem cells, and tumor cells. It may bring us a new approach for the resistance, recurrence, and metastasis of tumors.

Cellular immunotoxicity of rAAV gene medicine and possible solutions / 药学学报

Gene medicine based on recombinant adeno-associated virus (rAAV) vector has rapidly become the prior-choose reagent for gene therapy, since it had been shown that the rAAV was able to stably express many genes in vivo without detectable side-effect. However, recent findings of CTL immune responses to AAV capsid in a clinical trial highlighted a new issue regarding safety that previously was not identified in animal studies. Obviously it is so important to understand the interaction of rAAV with the immune system in details for the safety and success of rAAV gene medicine. In this review we evaluate several current hypotheses aiming to explain the cellular immunotoxicity, also analysis the current findings including the presentation kinetics of the capsid antigen and the activation of CTL. Focusing on the key steps of the immune response several solutions are proposed, including immunosuppression, optimization of vector and improvement of purity, in order to insure clinical safety and efficacy of rAAV.

Oral recombinant adeno-associated virus gene medicine / 药学学报

The efficacy of recombinant adeno-associated virus (rAAV) vector-mediated gene delivery to the gastrointestinal tract has been paid a considerable attention over the last 10 years, since our first report on the oral gene pill strategy in Nature Medicine, even though there are still several potential obstacles for this route to overcome in order to obtain efficient gene delivery. The preclinical results of oral rAAV gene medicine are summarized in this review, and special attention is paid on its pharmacokinetic and pharmacodynamic aspects with an emphasis on drug delivery, absorption, distribution and transduction. The rAAV based vectors have been shown promising results in human clinical trials with fewer safety concerns over other gene medicines. However, the underlying mechanisms and biopharmaceutical features of oral rAAV gene medicine remain to be explored extensively and intensively to develop this novel technology as a treatment for a wider range of diseases.