Adjuvant screening of the Senecavirus A inactivated vaccine in mice and evaluation of its immunogenicity in pigs.

BACKGROUND: Senecavirus A (SVA) causes an emerging vesicular disease (VD) with clinical symptoms indistinguishable from other vesicular diseases, including vesicular stomatitis (VS), foot-and-mouth disease (FMD), and swine vesicular disease (SVD). Currently, SVA outbreaks have been reported in Canada, the U.S.A, Brazil, Thailand, Vietnam, Colombia, and China. Based on the experience of prevention and control of FMDV, vaccines are the best means to prevent SVA transmission. RESULTS: After preparing an SVA inactivated vaccine (CH-GX-01-2019), we evaluated the immunogenicity of the SVA inactivated vaccine mixed with Imject® Alum (SVA + AL) or Montanide ISA 201 (SVA + 201) adjuvant in mice, as well as the immunogenicity of the SVA inactivated vaccine combined with Montanide ISA 201 adjuvant in post-weaned pigs. The results of the mouse experiment showed that the immune effects in the SVA + 201 group were superior to that in the SVA + AL group. Results from pigs immunized with SVA inactivated vaccine combined with Montanide ISA 201 showed that the immune effects were largely consistent between the SVA-H group (200 µg) and SVA-L group (50 µg); the viral load in tissues and blood was significantly reduced and no clinical symptoms occurred in the vaccinated pigs. CONCLUSIONS: Montanide ISA 201 is a better adjuvant choice than the Imject® Alum adjuvant in the SVA inactivated vaccine preparation, and the CH-GX-01-2019 SVA inactivated vaccine can provide effective protection for pigs.

Platycodin D2 enhances P21/CyclinA2-mediated senescence of HCC cells by regulating NIX-induced mitophagy.

BACKGROUND: Hepatocellular carcinoma (HCC) cells usually show strong resistance to chemotherapy, which not only reduces the efficacy of chemotherapy but also increases the side effects. Regulation of autophagy plays an important role in tumor treatment. Cell senescence is also an important anti-cancer mechanism, which has become an important target for tumor treatment. Therefore, it is of great clinical significance to find anti-HCC drugs that act through this new mechanism. Platycodin D2 (PD2) is a new saponin compound extracted from the traditional Chinese medicine Platycodon grandiflorum. PURPOSE: Our study aimed to explore the effects of PD2 on HCC and identify the underlying mechanisms. METHODS: First, the CCK8 assay was used to detect the inhibitory effect of PD2 on HCC cells. Then, different pathways of programmed cell death and cell cycle regulators were measured. In addition, we assessed the effects of PD2 on the autophagy and senescence of HCC cells by flow cytometry, immunofluorescence staining, and Western blotting. Finally, we studied the in vivo effect of PD2 on HCC cells by using a mouse tumor-bearing model. RESULTS: Studies have shown that PD2 has a good anti-tumor effect, but the specific molecular mechanism has not been clarified. In this study, we found that PD2 has no obvious toxic effect on normal hepatocytes, but it can significantly inhibit the proliferation of HCC cells, induce mitochondrial dysfunction, enhance autophagy and cell senescence, upregulate NIX and P21, and downregulate CyclinA2. Gene silencing and overexpression indicated that PD2 induced mitophagy in HCC cells through NIX, thereby activating the P21/CyclinA2 pathway and promoting cell senescence. CONCLUSIONS: These results indicate that PD2 induces HCC cell death through autophagy and aging. Our findings provide a new strategy for treating HCC.

[Construction, screening and immunogenicity of the recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2].

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.

Construction and immunogenicity of Senecavirus A virus-like particle vaccine with adjuvant.

Senecavirus A (SVA) is constantly associated with vesicular disease in pigs, and the clinical symptoms of pig infection with SVA are indistinguishable from other porcine vesicular diseases. Vaccine is one of the best methods to eliminate and control the spread of SVA. Virus-like particles (VLPs) can play important roles in prevention for infectious diseases. Here, the SVA VLPs was assembled by the baculovirus expression vector system, and the immunogenicity of the SVA VLPs mixed with different adjuvants were evaluated in mice and pigs. Two recombinant baculoviruses (rPFBD-VP1-VP3 and rPFBD-VP2-VP4) were constructed, which co-infected with Sf9 suspension cells to assemble SVA VLPs successfully. SVA VLPs mixed with ISA201 adjuvant and ISA201 +Poly(I:C) adjuvant produced higher levels of neutralizing antibody, specific antibody (total IgG, IgG1, IgG2a and IgG2b) and cytokines in the T cells. And there was no significant difference between SVA VLPs+ 201 group and SVA VLPs+Poly(I:C)+ 201 group. Pigs immunized with high dose of SVA VLPs mixed with ISA201 adjuvant could produce higher titers of neutralizing antibody and SVA-specific antibody. Furthermore, the protection rates of SVA VLPs-H and SVA VLPs-L were 100% and 80%, and the viral load of SVA VLPs-H group is the lowest in all SVA VLPs groups. It is the first time to develop the SVA VLPs using the baculovirus expression vector system, which may lay the foundation for the research and development of SVA vaccine.

Licochalcone A Induces Ferroptosis in Hepatocellular Carcinoma via Reactive Oxygen Species Activated by the SLC7A11/GPX4 Pathway.

Liver cancer is a common malignant tumor, and its incidence is increasing yearly. Millions of people suffer from liver cancer annually, which has a serious impact on global public health security. Licochalcone A (Lico A), an important component of the traditional Chinese herb licorice, is a natural small molecule drug with multiple pharmacological activities. In this study, we evaluated the inhibitory effects of Lico A on hepatocellular carcinoma cell lines (HepG2 and Huh-7), and explored the inhibitory mechanism of Lico A on hepatocellular carcinoma. First, we evaluated the inhibitory effects of Lico A on hepatocellular carcinoma, and showed that Lico A significantly inhibited and killed HepG2 and Huh-7 cells in vivo and in vitro. Transcriptomic analysis showed that Lico A inhibited the expression of solute carrier family 7 member 11 (SLC7A11), which induced ferroptosis. We confirmed through in vivo and in vitro experiments that Lico A promoted ferroptosis in hepatocellular carcinoma cells by downregulating SLC7A11 expression, thereby inhibiting the glutathione (GSH)-glutathione peroxidase 4 (GPX4) pathway and inducing activation of reactive oxygen species (ROS). In this study, we suggest that Lico A is a potential SLC7A11 inhibitor that induces ferroptotic death in hepatocellular carcinoma cells, thereby providing a theoretical basis for the development of natural small molecule drugs against hepatocellular carcinoma.

Is remnant preservation in anterior cruciate ligament reconstruction superior to the standard technique? An overview of systematic reviews.

BACKGROUND: Anterior cruciate ligament injury is a common knee joint injury. Anterior cruciate ligament reconstruction is a common surgical treatment to treat anterior cruciate ligament injury. It may have certain advantages to retain the ligament stump during the operation, but the results of systematic evaluation on whether to retain the ligament stump are different. The conclusion is still controversial, and the quality needs to be strictly evaluated. OBJECTIVE: To evaluate the methodological quality, risk of bias, reporting quality and evidence quality of the systematic review of remnant preservation in anterior cruciate ligament reconstruction, and to provide reference for clinical work. METHODS: We systematically searched the system evaluations in 8 electronic databases, the languages were limited to Chinese and English, and the time limit was from the establishment of the database to June 2023. Two reviewers independently screened literature and extracted data. The methodological quality, risk of bias, reporting quality and quality of evidence were evaluated by AMSTAR-2, ROBIS, PRISMA and GRADE tools. RESULTS: A total of 14 systematic reviews were included. The evaluation of results showed that the methodological quality of the included systematic reviews was relatively low, of which 5 were low quality and 9 were critically low quality. A small number of systematic reviews were low risk of bias. The system evaluation reports are relatively complete, but the lack of program registration is a common problem. A total of 111 pieces of clinical evidence were extracted from the included 14 systematic reviews. The quality of evidence was generally low, with only 7 pieces of high-quality evidence, 45 pieces of medium-quality evidence, and the rest were low and very low-quality evidence. Among the reasons for relegation, imprecision is the most common, followed by inconsistency and indirectness. The existing evidence shows that patients after anterior cruciate ligament reconstruction with remnant preservation have certain advantages in knee joint function, joint stability and proprioception recovery, which may be a more effective surgical method. However, it may also increase the incidence of postoperative complications and adverse reactions. CONCLUSION: Compared with Standard Technique, Remnant Preservation in Anterior Cruciate Ligament Reconstruction has more advantages in restoring joint function and stability and proprioception. But the potential risks should also be considered by surgeons. At present, the quality of evidence is generally low, and the reliability of the conclusion is insufficient. It still needs to be verified and further in-depth research is needed.

Peimisine ameliorates DSS-induced colitis by suppressing Jak-Stat activation and alleviating gut microbiota dysbiosis in mice.

OBJECTIVES: Inflammatory cytokine secretion and gut microbiota dysbiosis play crucial roles in ulcerative colitis. In this research, the protective effects of peimisine on colitis mice were investigated. METHODS: The protective effects were evaluated by the disease activity index, colonic length, hematoxylin-eosin, and AB/PAS Staining. The protective mechanisms were analyzed by ELISA, Western-blot, immunohistochemistry staining, immunofluorescence staining, and 16S rRNA gene analysis. KEY FINDINGS: The results showed that peimisine treatment could reduce the disease activity index, prevent colonic shortening, and alleviate colon tissue damage. Peimisine treatment also decreased the levels of MCP-1, IL-1ß, IL-6, IFN-γ, TNF-α and affected macrophage polarization and Th17/Treg cell balance by downregulating the expression of jak1/2, p-jak1/2, stat1/3, and p-stat1/3. Moreover, peimisine treatment significantly increased the abundances of beneficial microbes (e.g. Ruminococcaceae UCG-014 and Lachnospiraceae_NK4A136_group) and decreased the abundances of harmful microbes (e.g. Bacteroides and Escherichia). CONCLUSIONS: Peimisine can ameliorate colitis by inhibiting Jak-Stat signaling pathway, reversing gut microbiota alterations, suppressing macrophage M1 polarization, maintaining the Th17/Treg cell balance, and reducing sustained inflammatory cytokines-related inflammatory injury.

Human adenovirus type 7 subunit vaccine induces dendritic cell maturation through the TLR4/NF-κB pathway is highly immunogenic.

Introduction: Human adenovirus type 7 (HAdv-7) infection is the main cause of upper respiratory tract infection, bronchitis and pneumonia in children. At present, there are no anti- adenovirus drugs or preventive vaccines in the market. Therefore, it is necessary to develop a safe and effective anti-adenovirus type 7 vaccine. Methods: In this study, In this study, we used the baculovirus-insect cell expression system to design a recombinant subunit vaccine expressing adenovirus type 7 hexon protein (rBV-hexon) to induce high-level humoral and cellular immune responses. To evaluate the effectiveness of the vaccine, we first detected the expression of molecular markers on the surface of antigen presenting cells and the secretion of proinflammatory cytokines in vitro. We then measured the levels of neutralizing antibodies and T cell activation in vivo. Results: The results showed that the rBV-hexon recombinant subunit vaccine could promote DC maturation and improve its antigen uptake capability, including the TLR4/NF-κB pathway which upregulated the expression of MHCI, CD80, CD86 and cytokines. The vaccine also triggered a strong neutralizing antibody and cellular immune response, and activated T lymphocytes. Discussion: Therefore, the recombinant subunit vaccine rBV-hexon promoted promotes humoral and cellular immune responses, thereby has the potential to become a vaccine against HAdv-7.

Neobavaisoflavone induces pyroptosis of liver cancer cells via Tom20 sensing the activated ROS signal.

BACKGROUND: Neobavaisoflavone (NBIF), a natural active ingredient isolated from Psoralea, possesses anti-inflammatory, anti-cancer, and antioxidant properties; however, the anti-tumor mechanism of NBIF has not been thoroughly investigated, and the inhibitory effect and inhibitory pathway of NBIF on liver cancer are still unknown. PURPOSE: Our study aimed to explore the effects of NBIF on hepatocellular carcinoma and its potential mechanisms. METHODS: First, we detected the inhibition of NBIF on HCC cells by the CCK8 assay and then observed the morphological changes of the cells under the microscope. Besides, we analyzed the changes in the pyroptosis level of NBIF when inhibiting the cells through flow cytometry, immunofluorescence, and a western blot assay. Finally, we used a mouse tumor-bearing model to explore the effects of NBIF in vivo on HCCLM3 cells. RESULTS: NBIF-treated HCC cells exhibited specific features of pyroptosis. Analysis of pyroptosis-related protein levels revealed that NBIF primarily induced pyroptosis in HCC cells via the caspase-3-GSDME signaling pathway. Then, we demonstrated that NBIF impacted the protein expression of Tom20 by producing ROS in HCC cells, hence promoting the recruitment of Bax to mitochondria, activating caspase-3, cutting GSDME, and triggering pyroptosis. CONCLUSIONS: By activating ROS, NBIF was able to trigger pyroptosis in HCC cells, providing an experimental basis for the future study of new treatments for liver cancer.

Inhibitory Effects of Esculetin on Liver Cancer Through Triggering NCOA4 Pathway-Mediation Ferritinophagy in vivo and in vitro.

Objective: To explore the effects of Esculetin on liver cancer and explore potential mechanisms of Esculetin-inducing cells death. Methods: Esculetin's effects on the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells were detected by using CCK8, crystal violet staining, wound healing, TranswellTM and Annexin V-FITC/PI. Flow cytometry, fluorescence staining, Western blot, T-AOC, DPPH radical scavenging assay, hydroxyl radical's inhibitory capability and GSH test were used to examine the esculetin's effects on the ROS level, the oxidation-related substances and proteins' expression in hepatoma cells. In vivo experiment was performed by xenograft model. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by esculetin. Live cell probe, Western blot, Fe2+ content, MDA, HE staining, Prussian blue staining and immunohistochemistry were used to examine the ferritinophagy-related phenomenon induced by esculetin in hepatoma cells. The relationship between esculetin and NCOA4-mediated ferritinophagy was confirmed through gene silence and overexpression, immunofluorescence staining and Western blot. Results: Esculetin suppressed the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells significantly, influenced the oxidative stress level, altered the autophagy and iron metabolism levels in cells, and produced a ferritinophagy-related phenomena. Esculetin increased the levels of cellular lipid peroxidation and reactive oxygen species. In vivo, esculetin could decrease tumour volume, promote LC3 and NCOA4 expressions, suppresse hydroxyl radical's inhibiting capacity and GSH, increase Fe2+ and MDA levels, decrease antioxidant proteins expression in tumour tissue. In addition, Esculetin could also increase the iron deposition of tumour tissues, promote ferritinophagy, and induce tumours' ferroptosis. Conclusion: Esculetin has an inhibitory effect on liver cancer in vivo and in vitro through triggering NCOA4 pathway-mediation ferritinophagy.

Fu-Zheng-Xuan-Fei formula promotes macrophage polarization and Th17/Treg cell homeostasis against the influenza B virus (Victoria strain) infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu-Zheng-Xuan-Fei formula (FF) is a prescription that has been clinically used through the basic theory of traditional Chinese medicine (TCM) for treating viral pneumonia. Although FF possesses a prominent clinical therapeutic effect, seldom pharmacological studies have been reported on its anti-influenza B virus (IBV) activity. AIM OF THE STUDY: Influenza is an acute infectious respiratory disease caused by the influenza virus, which has high annual morbidity and mortality worldwide. With a global decline in the COVID-19 control, the infection rate of influenza virus is gradually increasing. Therefore, it is of great importance to develop novel drugs for the effective treatment of influenza virus. Apart from conventional antiviral drugs, TCM has been widely used in the clinical treatment of influenza in China. Therefore, studying the antiviral mechanism of TCM can facilitate the scientific development of TCM. MATERIALS AND METHODS: Madin-Darby canine kidney cells (MDCK) and BALB/c mice were infected with IBV, and FF was added to evaluate the anti-IBV effects of FF both in vitro and in vivo by Western blotting, immunofluorescence, flow cytometry, and pathological assessment. RESULTS: It was found that FF exhibited anti-viral activity against IBV infection both in vivo and in vitro, while inducing macrophage activation and promoting M1 macrophage polarization. In addition, FF effectively regulated the signal transducer and activator of transcription (STAT) signaling pathway-mediated Th17/Treg balance to improve the lung tissue damage caused by IBV infection-induced inflammation. The findings provided the scientific basis for the antiviral mechanism of FF against IBV infection. CONCLUSIONS: This study shows that FF is a potentially effective antiviral drug against IBV infection.

LncRNA ZFAS1 protects chondrocytes from IL-1ß-induced apoptosis and extracellular matrix degradation via regulating miR-7-5p/FLRT2 axis.

BACKGROUND: Increasing evidence suggested that long non-coding RNAs (lncRNAs) played vital roles in osteoarthritis (OA) progression. In this study, we aimed to reveal the protective roles of lncRNA ZFAS1 in osteoarthritis (OA) and further investigated its underlying mechanism. METHODS: The chondrocytes were stimulated by IL-1ß to establish an in vitro OA model. Then, the expression of ZFAS1, miR-7-5p, and FLRT2 in chondrocytes was determined by qRT-PCR. Gain- and loss-of-function assays of ZFAS1, miR-7-5p and FLRT2 were conducted. CCK-8 assay and flow cytometry analysis were performed to detect cell viability and apoptosis rate. The expression levels of cartilage-related proteins, including MMP13, ADAMTS5, Collagen II, and Aggrecan, were measured by western blot analysis. The interaction between ZFAS1 and miR-7-5p, as well as miR-7-5p and FLRT2, was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: The expression of ZFAS1 and FLRT2 was down-regulated, while the expression of miR-7-5p was up-regulated in chondrocytes exposed to IL-1ß. ZFAS1 overexpression promoted cell viability and suppressed apoptosis in IL-1ß-treated chondrocytes. Besides, ZFAS1 overexpression suppressed the expression of MMP13 and ADAMTS5, but promoted the expression of Collagen II and Aggrecan to suppress ECM degradation. The mechanistic study showed that ZFAS1 sponged miR-7-5p to regulate FLRT2 expression. Furthermore, the overexpression of miR-7-5p could neutralize the effect of ZFAS1 in IL-1ß-treated chondrocytes, and suppression of FLRT2 counteracted the miR-7-5p down-regulation role in IL-1ß-treated chondrocytes. CONCLUSIONS: ZFAS1 could promote cell viability of IL-1ß-treated chondrocytes via regulating miR-7-5p/FLRT2 axis. Trial registration Not applicable.

Human adenovirus type 7 virus-like particle vaccine induces Dendritic cell maturation through the TLR4/NF-κB pathway and is highly immunogenic.

Human adenovirus type 7 (HAdv-7) infection is the main cause of upper respiratory tract infection, bronchitis and pneumonia in children. At present, there are no anti-adenovirus drugs or preventive vaccines in the market. Therefore, it is necessary to develop a safe and effective anti-adenovirus type 7 vaccine. In this study, we designed a virus-like particle vaccine expressing the epitopes of hexon and penton of adenovirus type 7 with hepatitis B core protein (HBc) as the vector to induce high-level humoral and cellular immune responses. To evaluate the effectiveness of the vaccine, we first detected the expression of molecular markers on the surface of antigen presenting cells and the secretion of proinflammatory cytokines in vitro. We then measured the levels of neutralizing antibodies and T cell activation in vivo. The results showed that the HAdv-7 virus-like particles (VLPs) recombinant subunit vaccine could activate the innate immune response, including the TLR4/NF-κB pathway which upregulated the expression of MHC II, CD80, CD86, CD40 and cytokines. The vaccine also triggered a strong neutralizing antibody and cellular immune response and activated T lymphocytes. Therefore, the HAdv-7 VLPs promoted humoral and cellular immune responses, thereby potentially enhancing protection against HAdv-7 infection.

Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway.

BACKGROUND: Apoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. Herein, we found that Apoptin induced a strong and lasting endoplasmic reticulum (ER) stress response, Ca2+ imbalance, and triggered the mitochondrial apoptotic pathway. The aim of this study was to explore the mechanisms by which Apoptin exhibited anti-tumor effects in HepG-2 cells. METHODS: The intracellular levels of calcium (Ca2+ ) were induced by ER stress and determined by electron microscopy, flow cytometry, and fluorescence staining. The mitochondrial injury was determined by mitochondrial membrane potential and electron microscopy. Western blotting was used to investigate the levels of key proteins in ER stress and the apoptotic pathway in mitochondria. The relationship between Ca2+ levels and apoptosis in Apoptin-treated cells was analyzed using a Ca2+ chelator (BAPTA-AM), flow cytometry, and fluorescence staining. We also investigated the in vivo effects of Ca2+ imbalance on the mitochondrial apoptotic pathway using tumor tissues xenografted on nude mice. RESULTS: This study showed that Apoptin induced a strong and long- lasting ER stress and injury, which subsequently led to an imbalance of cellular Ca2+ levels, a reduction in the mitochondrial membrane potential, a significant extent image in the mitochondrial structure, and an increase in the expression levels of Smac/Diablo and Cyto-C. CONCLUSIONS: In summary, Apoptin induced apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway. This study provided a new direction for antitumor research in Apoptin.

Effects of glabridin on malignant biological behaviors of lung adenocarcinoma A549 cells and its molecular mechanism / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨光甘草定对肺腺癌细胞A549恶性生物学行的影响及其分子机制。方法：常规方法培养A549细胞和人正常肺上皮细胞BEAS-2B，用不同浓度的光甘草定和或顺铂对其进行处理，通过结晶紫染色、CCK-8法检测光甘草定、顺铂对A549、BEAS-2B细胞增殖活力的影响，Transwell小室实验、细胞划痕实验检测光甘草定对A549细胞侵袭、迁移能力的影响，流式细胞术检测光甘草定对A549细胞凋亡的影响，3D超低黏附板培养法培养A549细胞后采用CCK-8法检测光甘草定对A549细胞增殖的影响，WB法检测光甘草定对A549细胞中上皮间质转化（EMT）相关蛋白表达的影响；构建A549细胞移植瘤模型后检测光甘草定、顺铂对移植瘤的生长以及移植瘤组织中EMT相关蛋白表达的影响。结果：光甘草定、顺铂呈剂量依赖性地显著抑制A549细胞的增殖（P<0.05或P<0.01）、细胞迁移（P<0.05或P<0.01）和侵袭能力（P<0.05或P<0.01），光甘草定能诱导A549细胞凋亡（P<0.01），抑制A549细胞中N-cadherin、snail和vimentin蛋白的表达，促进E-cadherin蛋白表达；光甘草定、顺铂均能抑制A549移植瘤的生长，抑制移植瘤组织中Ki67、N-cadherin、snail和vimentin蛋白的表达、促进E-cadherin蛋白的表达。结论：光甘草定可通过抑制A549细胞的增殖、迁移和侵袭，诱导A549细胞凋亡，其机制可能与其抑制细胞的EMT进程而产生抑癌作用有关。

Neobavaisoflavone induces pyroptosis in hepatocellular carcinoma Huh-7 cells through caspase-3/GSDME signal pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨新补骨脂异黄酮（NBIF）对肝细胞癌（HCC）Huh-7细胞焦亡的影响及其分子机制。方法：体外培养Huh-7细胞，用CCK-8法检测不同浓度的NBIF处理48 h时对细胞存活率的影响，光学显微镜下观察NBIF处理后Huh-7细胞的形态变化，乳酸脱氢酶（LDH）释放实验检测细胞的LDH释放量，WB实验检测细胞中GSDME、caspase-3的蛋白水平变化。采用siRNA干扰Huh-7细胞中caspase-3、GSDME表达后，CCK-8法检测NBIF处理对细胞存活率的影响，WB实验检测GSDME蛋白表达水平，观察NBIF处理对细胞形态的影响，并检测细胞LDH释放量。结果：60 μmol/L以上的NBIF均能显著抑制Huh-7细胞的增殖（均P<0.01），光学显微镜下观察到NBIF处理后的细胞出现肿胀、吐泡现象，且LDH释放增加（P<0.01）；WB实验结果表明，NBIF能够激活caspase-3蛋白并切割GSDME蛋白，增加GSDME-N的表达（均P<0.01）。干扰caspase-3、GSDME表达后，NBIF对细胞的抑制作用减弱（均P<0.01），GSDME-N蛋白表达受到抑制（P<0.01），显微镜下细胞肿胀、吐泡现象几乎消失，LDH释放明显减少（P<0.05）。结论：NBIF能够通过caspase-3/GSDME途径诱导Huh-7细胞发生焦亡，从而抑制HCC细胞的增殖，为HCC的治疗提供一种新思路。

Apoptin inhibits glycolysis and regulates autophagy by targeting pyruvate kinase M2 (PKM2) in lung cancer A549 cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis, and which plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, that can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62 and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells, were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in energy metabolism of A549 cells.

Assessment of the immunogenicity and protection of a Nipah virus soluble G vaccine candidate in mice and pigs.

Nipah virus (NiV) is a newly emerged extremely dangerous zoonotic pathogen highly fatal to humans. Currently, no approved vaccine is available against NiV. This study employed a mammalian eukaryotic system to express NiV soluble G glycoprotein (NiV-sG), using CpG oligodeoxynucleotides (CpG)/Aluminum salt (Alum) as adjuvants to obtain a recombinant subunit vaccine candidate. We also evaluated the immunogenicity and efficacy of the protein in mice and pigs. The results showed that humoral and cellular immune responses were induced in all the vaccination groups in two animal models. The levels of specific and neutralizing antibodies and the proliferation levels of T helper(Th) cells were significantly higher than those in the control group. The protective efficacy of the subunit vaccines evaluated in the pseudovirus in vivo infection mouse model strongly suggested that this vaccine could provide protective immunity against NiV. A neoadjuvant (HTa) based on liposomes and cholera toxin combined with CpG/Alum was exploited and evaluated in mice. The neoadjuvant group showed a more protective efficacy than the CpG/Alum group. The aforementioned results indicated that the subunit vaccine could be used as a promising candidate vaccine for preventing Nipah virus infection.

Synergistic pathogenicity by coinfection and sequential infection with JXA1-like HP-PRRSV and PCV2d in PCV2 antibody-positive post-weaned pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus (PCV) are two important pathogens, which caused respiratory disease in pigs. PRRSV and PCV2 had caused great economic losses to the pig industry. Pigs coinfection with PCV2 and PRRSV were common in the clinic, PCV2 antibodies can be detected in most of the pigs. PCV2d and HP-PRRSV(JXA1-like) were two major viruses circulating in the pigs in China. In this study, HP-PRRSV (JXA1-like) and PCV2d were used to coinfect and (or) sequential infect 5-week-old weaned PCV2-antibody positive pigs and the clinical indications, pathological, virus load, and specific antibodies of the challenged post-weaned piglets were evaluated. Thirty 5-week-old post-weaned pigs were divided into six groups infected with PBS, PCV2, PRRSV, PCV2-PRRSV, PRRSV-PCV2, and Co-PRRSV-PCV2 according to the PCV2 specific antibodies. Pigs infected with PRRSV can experience diarrhea, increased body temperature, weight loss, and even death. The pigs in the PRRSV infected group and PRRSV-PCV2 infected group showed severe clinical symptoms, high mortality, and low average daily gain. The main pathological changes were widening of the lung interstitium, lung adhesion, and so on. The PRRSV-PCV2 infected group showed high levels of TNF-α and IL-2. In conclusion, PRRSV and PRRSV-PCV2 sequential infected pigs showed most pathogenic signs, and PCV2-PRRSV sequential infected pigs showed less pathogenicity than pigs of PCV2 and PRRSV coinfection and PRRSV monoinfection from day 10-14, partially suppressing the cytokine storm produced by PRRSV.

Recombinant Oncolytic Adenovirus Combined with Cyclophosphamide Induces Synergy in the Treatment of Breast Cancer in vitro and in vivo.

Purpose: Oncolytic virus therapy has gradually become an integral approach in cancer treatment. We explored the therapeutic effects of the combination of a dual cancer-selective anti-tumor recombinant adenovirus (Ad-Apoptin-hTERTp-E1a) and cyclophosphamide on breast cancer cells. Methods: The inhibition of MCF-7 and MDA-MB-231 breast cancer cells by Ad-Apoptin-hTERTp-E1a (Ad-VT), cyclophosphamide, and Ad-VT + Cyclophosphamide was investigated using the CCK-8 assay. The combination index (CI) was calculated using CalcuSyn software to determine the best combination based on the inhibition rates of the different treatment combinations. The CCK-8 assay and crystal violet staining were used to detect the cytotoxicity of the combined Ad-VT and cyclophosphamide in breast cancer cells and breast epithelial cells. Subsequently, Hoechst staining, annexin V flow cytometry, and JC-1 staining were used to analyze the inhibitory pathway of Ad-VT plus cyclophosphamide on breast cancer cells. Cell migration and invasion of breast cancer cells were assessed using the cell-scratch and Transwell assays. The anti-tumor effects of different treatment groups in a tumor-bearing nude mouse model also were analyzed. Results: The treatment combination of Ad-VT (40 MOI) and cyclophosphamide (400 µM) significantly inhibited MCF-7 and MDA-MB-231 cells and reduced the toxicity of cyclophosphamide in normal cells. Ad-VT primarily induced breast cancer cell apoptosis through the endogenous apoptotic pathway. Apoptosis was significantly increased after treatment with Ad-VT plus cyclophosphamide. The combination significantly inhibited the migration and invasion of MCF-7 and MDA-MB-231 cells. The in vivo experiments demonstrated that exposure to Ad-VT plus cyclophosphamide significantly inhibited tumor growth and extended the survival time of the nude mice. Conclusion: Ad-VT plus cyclophosphamide reduced toxicity and exhibited increased efficacy in treating breast cancer cells.