Activity of Baicalin Against Human Cytomegalovirus In Vitro and Its Effect on the Apoptosis of Human Embryo Lung Fibroblasts Infected with Human Cytomegalovirus
刘杨, 黄媛, 廖毅, 秦文卿, 刘玲玲, 李革, 舒赛男, 方峰
华中科技大学同济医学院附属同济医院儿科学系,武汉 430030
LIU Yang,, HUANG Yuan, LIAO Yi, QIN Wenqing, LIU Lingling, LI Ge, SHU Sainan, FANG Feng,
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Objective To elucidate activity of baicalin against human cytomegalovirus (HCMV) in vitro, and explore its effect on apoptosis of human embryo lung fibroblasts (HEL) infected with HCMV. Methods CCK-8 kit was used to determine the maximum tolerated dose (MTC) of HEL cell to baicalin while the anti-HCMV median efficacious concentration (EC50) of baicalin was determined by standard plaque reduction method. After treatment with baicalin of different concentrations for 24, 48, 72 and 96 h, cell apoptosis and pro-caspase-3 expression was detected by flow cytometry and Western blotting, respectively. Results The MTC of baicalin was 20.6 μg·mL-1; The EC50 of anti-HCMV of baicalin was 16.13 μg·mL-1; The apoptosis rate increased gradually in the groups with low and high multiplicity HCMV infection at early time, showing significant dose-dependent manner. While the ratio of apoptotic cells was going to decrease in high multiplicity infection group 96 h after the infection. The expression of pro-caspase-3 was significantly higher in high-dose baicalin treatment group than in the infection control group (P<0.05). Conclusion Baicalin has a direct anti-HCMV effect in vitro. One of the mechanisms might be related with it inhibiting cell apoptosis and antagonizing activation of pro-caspase-3.
Key words:
Baicalin
;
Human cytomegalovirus
;
Human embryo lung fibroblast
;
Apoptosis
;
Pro-caspase-3 expression
人巨细胞病毒(human cytomegalovirus,HCMV)是一种世界范围内普遍感染的双链DNA病毒,一旦感染将终身携带。我国是HCMV感染的高发地区,人群抗HCMV-IgG阳性率高,为86%~96%[1]。HCMV感染对胎儿、婴幼儿、器官移植患者、艾滋病患者等免疫低下人群可造成严重损害,甚至危及生命。但是,一直以来,抗HCMV感染药物的研究进展缓慢,能用于临床的抗HCMV药物寥寥无几,故寻找和开发安全、副作用小而且疗效佳的抗HCMV药物一直是医药学界的努力方向和研究任务。黄芩具有泻火解毒、清热燥湿等功效。国内外学者已经发现,黄芩提取物黄芩苷可以抑制流感病毒、登革热病毒、单纯疱疹病毒-Ⅰ型(herpes simplex virus type 1,HSV-Ⅰ)、HSV-Ⅱ等多种病毒的复制[2-5]。本课题组前期在黄芩苷抗小鼠巨细胞病毒(murine cytomegalovirus,MCMV)的整体研究中发现,黄芩苷对MCMV具有直接抑制作用,可显著下调MCMV感染小鼠唾液腺病毒滴度,减轻MCMV感染小鼠的肝脏病变[6]。因巨细胞病毒具有严格的宿主种属特异性,故考虑黄芩苷是否对HCMV也具有同样的抗病毒活性,抗病毒作用机制如何,笔者尚未见研究报道。调控细胞凋亡是HCMV致病的重要机制之一。本课题组前期研究发现,HCMV肝炎患儿,肝组织细胞凋亡率为53.72%,显著高于正常儿童的肝细胞凋亡率7.19%[7]。DOU等[8]发现,HCMV感染造成的血小板减少症也是通过诱导产板巨核细胞祖细胞的过度凋亡造成。本课题以HCMV感染的人胚肺成纤维细胞(human embryo lung fibroblast,HEL)为体外模型,检测黄芩苷体外抗HCMV的活性,并从细胞凋亡的角度探讨黄芩苷抗HCMV可能作用机制。
Fig.1
Effect of baicalin on apoptosis ratio of HEL with low multiplicity infection(x¯±s,n=5) Compared with low multiplicity infection group, *1P<0.05; Compared with low multiplicity infection and low dose baicalin group, *2P<0.05
Fig.2
Effect of baicalin on apoptosis ratio of HEL with high multiplicity infection(x¯±s,n=5) Compared with high multiplicity infection group, *1P<0.05
图3
4组72 h pro-caspase-3表达量 A.模拟感染组;B.高感染复数组;C.高感染+小剂量黄芩苷组;D.高感染+大剂量黄芩苷组
Fig.3
Pro-caspase-3 expression of four groups at 72 h A. mimetic infection group; B. high multiplicity infection group; C. high multiplicity infection and low dose baicalin group; D. high multiplicity infection and high dose baicalin group
Fig.4
Adjusted value κ of pro-caspase-3 level in four groups(x¯±s,n=5) A. mimetic infection group; B. high multiplicity infection group; C. high multiplicity infection and low dose baicalin group; D. high multiplicity infection and high dose baicalin group. Compared with mimetic infection group, *1P<0.01; Compared with high multiplicity infection group, *2P<0.05
XUG,DOUJ,ZHANGL,et al.Inhibitory effects of baica-lein on the influenza virus in vivo is determined by baicalin in the serum[J].,2010,33(2):238-243.
Baicalein, an extract from Scutellaria baicalensis, was evaluated for its ability to inhibit the influenza virus in vivo. Oral administration of baicalein to BALB/c mice infected with the influenza A/FM1/1/47(H1N1) virus showed significant effects in preventing death, increasing the mean time to death, inhibiting lung consolidation, and reducing the lung virus titer in a dose-dependent manner. These effects are believed to be due to baicalin, the metabolite of baicalein in the serum. At a concentration of baicalin 2 microg/ml in overlay medium, it showed significant inhibition in the plaque assay, and the mean IC(50) value of baicalin was calculated as 1.2 microg/ml in the cytopathic effect assay. Our results showed that baicalein warrants further research as a potential antiinfluenza viral agent.
DOUJ,CHENL,XUG,et al.Effects of baicalein on Sendai virus in vivo are linked to serum baicalin and its inhibition of hemagglutinin-neuraminidase[J].,2011,156(5):793-801.
Parainfluenza viruses are significant respiratory-tract pathogens that are notorious for infecting children. However, there are no clinical drugs to control the infection caused by these viruses. Sendai virus (SeV) belongs to the family Paramyxoviridae and causes fatal pneumonia in mice, its natural host. Baicalein is a flavonoid derived from the root of Scutellaria baicalensis, which is a traditional Chinese medicine that has been used for hundreds of years and has demonstrated a variety of biological activities. Our findings reveal that oral administration of baicalein to mice infected with Sendai virus results in a significant reduction in virus titers in the lungs and protection from death. The in vivo inhibitory effects of baicalein on Sendai virus are determined by baicalin in the serum. The mean IC(50) of baicalin was 0.71 μg/ml in an HA inhibition assay and 3.22 μg/ml in an NA inhibition assay. The mean IC(50) of baicalin in a CPE assay was measured to be 0.70 μg/ml, and significant inhibition was observed in a plaque assay at a concentration of 1.6 μg/ml baicalin in overlay medium, which suggests that baicalein is a potential anti-parainfluenzaviral agent in vivo.
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DOUJ,LIX,CAIY,et al.Human cytomegalovirus induces caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by activating the JNK pathway[J].,2010,91(4):620-629.
Human cytomegalovirus (HCMV) infection is usually implicated in thrombocytopenia occurring in newborns and immunocompromised patients. However, the underlying mechanisms remain elusive. This study was conducted to investigate the effects of HCMV infection on the viability of megakaryocytic CHRF-288-11 cells and the underlying mechanisms involved. RT-PCR for determining mRNA expression of HCMV immediate early gene 1 and Western blot for measuring protein expression of late HCMV gene pp65 showed that CHRF-288-11 cells were susceptible to HCMV infection. HCMV infection reduced the viability of CHRF-288-11 cells via apoptosis in a dose- and time-dependent manner. Both caspase 3 and c-Jun terminal kinase (JNK) signaling pathway were activated in the HCMV-treated CHRF-288-11 cells. z-DEVD-fmk (a caspase inhibitor) and SP600125 (a JNK inhibitor) significantly prevented the death of CHRF-288-11 cells induced by HCMV, respectively. Furthermore, inhibition of JNK activity could reduce the formation of active caspase 3 induced by HCMV. Interestingly, the co-application of antivirus drug ganciclovir and SP600125 synergistically prevented the death of CHRF-288-11 cells induced by HCMV. Collectively, these findings suggest that HCMV infection may induce the caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by the activation of JNK signaling pathway.
YOSHIDAH,SUMICHIKAH,HAMANOS,et al.Induction of apoptosis of T cells by infecting mice with murine cytomegalovirus[J].,1995,69(8):4769-4775.
Abstract Cytomegalovirus (CMV) is associated with several lymphocyte dysfunctions, but the precise mechanisms of the dysfunctions are still unclear. To elucidate the mechanisms, a cell cycle-DNA content analysis was performed on splenic T cells of murine CMV (MCMV)-infected BALB/c mice. T cells from mice infected with 3 x 10(3) PFU of MCMV contained a higher percentage of hypodiploid nuclei after 12 or 24 h of culture than those from naive mice. T cells from infected mice also contained a larger amount of fragmented DNA. Taken together, these results suggested that infection with MCMV induced the apoptotic cell death of T cells. This induction of apoptosis accounted for the dysfunction of lymphocytes, at least partially. Flow cytometric analysis showed that T cells as well as B cells from MCMV-infected mice expressed an augmented level of Fas antigen, an apoptosis-associated cell surface molecule, which might be the cause of the apoptosis of cells. T cells from MCMV-infected C57BL/6-lpr/lpr mice with mutations at the lpr/fas locus, however, also showed a substantial level of apoptosis, which was reproducibly lower than that seen in C57BL/6 mice. Therefore, it was suggested that the Fas-mediated pathway contributed to but was not sufficient for the induction of apoptosis and that mechanisms other than the Fas-associated pathway were also involved in the induction of apoptosis.
BRUNEW.Inhibition of programmed cell death by cytome-galoviruses[J].,2011,157(2):144-150.
The elimination of infected cells by programmed cell death (PCD) is one of the most ancestral defense mechanisms against infectious agents. This mechanism should be most effective against intracellular parasites, such as viruses, which depend on the host cell for their replication. However, even large and slowly replicating viruses like the cytomegaloviruses (CMVs) can prevail and persist in face of cellular suicide programs and other innate defense mechanisms. During evolution, these viruses have developed an impressive set of countermeasures against premature demise of the host cell. In the last decade, several genes encoding suppressors of apoptosis and necrosis have been identified in the genomes of human and murine CMV (HCMV and MCMV). Curiously, most of the gene products are not homologous to cellular antiapoptotic proteins, suggesting that the CMVs did not capture the genes from the host cell genome. This review summarizes our current understanding of how the CMVs suppress PCD and which signaling pathways they target.
PATTERSON CE,SHENKT.Human cytomegalovirus UL36 protein is dispensable for viral replication in cultured cells[J].,1999,73(9):7126-7131.
Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.
MAVINAKERE MS,COLBERG-POLEY A M.Dual target-ing of the human cytomegalovirus UL37 exon 1 protein during permissive infection[J].,2004,85(Pt 2):323-329.
The human cytomegalovirus (HCMV) UL37 immediate-early (IE) gene minimally encodes three protein isoforms that share NH(2)-terminal sequences. The predominant UL37 isoform detected during HCMV infection was the UL37 exon 1 protein (pUL37x1), which was produced from IE and, more abundantly, through late times of infection. pUL37x1 was localized in both the endoplasmic reticulum (ER) and mitochondria in infected cells. To determine which UL37x1 NH(2)-terminal residues serve as ER and mitochondrial targeting signals, we examined the subcellular localization of two deletion mutants. pUL37x1Delta2-23, which lacks the hydrophobic leader, is neither translocated into the ER nor imported mitochondrially; conversely, pUL37x1Delta23-34, lacking the juxtaposed basic residues, was translocated into the ER but only imported weakly into mitochondria. These studies show for the first time the temporal production and localization of pUL37x1 during HCMV infection. The trafficking patterns of mutants suggest that the pUL37x1 targeting signal to ER and mitochondria is bipartite.
EVERS DL,CHAO CF,WANGX,et al.Human cytome-galovirus-inhibitory flavonoids:studies on antiviral activity and mechanism of action[J].,2005,68(3):124-134.
We report antiviral activity against human cytomegalovirus for certain dietary flavonoids and their likely biochemical mechanisms of action. Nine out of ten evaluated flavonoids blocked HCMV replication at concentrations that were significantly lower than those producing cytotoxicity against growing or stationary phase host cells. Baicalein was the most potent inhibitor in this series (IC 50 02=020.4–1.202μM), including positive control ganciclovir. Baicalein and genistein were chosen as model compounds to study the antiviral mechanism(s) of action for this series. Both flavonoids significantly reduced the levels of HCMV early and late proteins, as well as viral DNA synthesis. Baicalein reduced the levels of HCMV immediate-early proteins to nearly background levels while genistein did not. The antiviral effects of genistein, but not baicalein, were fully reversible in cell culture. Pre-incubation of concentrated virus stocks with either flavonoid did not inhibit HCMV replication, suggesting that baicalein did not directly inactivate virus particles. Baicalein functionally blocked epidermal growth factor receptor tyrosine kinase activity and HCMV nuclear translocation, while genistein did not. At 2402h post infection HCMV-infected cells treated with genistein continued to express immediate-early proteins and efficiently phosphorylate IE1-72. However, HCMV induction of NF-κB and increases in the levels of cell cycle regulatory proteins—events that are associated with immediate-early protein functioning – were absent. The data suggested that the primary mechanism of action for baicalein may be to block HCMV infection at entry while the primary mechanism of action for genistein may be to block HCMV immediate-early protein functioning.