| Background Enterovirus D68(EV-D68)is a respiratory virus of the genus Enterovirus and the picornavirus family and is a deadly pathogen.The susceptible population of the virus is a child that can cause many respiratory diseases and a range of diseases related to the nervous system.Unfortunately,there are currently no efficient drugs and vaccines to treat and prevent diseases caused by EV-D68.Many studies have shown that most of the virus replication is related to the cell cycle,Coxsackie virus A16(CA16)and Enterovirus 71(EV-A71)promote their own replication by inducing S phase arrest in host cells,while Enterovirus 68(EV-D68)promotes its own replication by inducing host cell G0/G1 arrest.The above indicates that the cycle arrest is conducive to viral replication,suggesting that we can use the relevant drugs to change the state of the host cell cycle,thereby changing the virus-dominated cycle system.Therefore,we hypothesized that the use of drugs that induce S-phase or G2/M arrest may be a new strategy for the treatment of EV-D68-related diseases in EV-D68,which induces G0/G1 arrest in host cells.What's more,there are no documents and reports on the results of this strategy so far.Objectives In the study,we investigated and studied the involvement of cell cycle regulation,especially the induction of S-phase arrest and induction of G2/M,based on the basis of EV-D68,which induces self-replication by host cell arrest in G0/G1 phase.The effect of Chinese medicine compounds with retardation on the replication of EV-D68 is intended to screen out the types of traditional Chinese medicine compounds that are beneficial for controlling EV-D68 replication and to explain its inhibitory mechanism.Methods In this study,human rhabdomyoblastoma RD was used as the research object,according to the influence of heavy traditional Chinese medicine compounds on cell cycle arrest,they were divided into two categories,and the effects of two kinds of traditional Chinese medicine compounds on EV-D68 replication were verified by various experimental methods.We found that emodin,artemisinin,and astragaloside induced S phase arrest,and that Pseudolaric acid B,Oridonin and Erianin induced G2/M phase arrest.First,the optimal concentration of the six drugs for the experiment was screened by the cell viability assay(MTT);Second,the cell cycle curve of the host cells was determined by Flow Cyto Metry,and it was used to verify the cell cycle arrest after virus infection and drug treatment.At the same time,the effects of viral infection on the cell cycle state of EV-D68 after treatment with two kinds of drugs were analyzed,and different cells were calculated.The number of cells in the cycle phase as a percentage of total cells.Thirdly,we used the Western Blot to quantitatively detect the relevant cyclins to explore whether the drug changes the cell cycle by regulating which cyclins.The cytopathic effect caused by virus and the protective effect on cells after drug treatment were observed by cell morphology and nuclear staining.The level of viral m RNA in cells was detected by RT-q PCR;the number of progeny virus was determined by titration(TCTD50/ml).The viral protein VP1 in the G2/M drug treatment group was detected by Western Blot;In addition,the virus released into the supernatant of the G2/M drug treatment group was tested for viral m RNA levels by RT-q PCR.Western Blot assay was used to evaluate the expression of viral VP1 protein.At the end of the experiment,the method of sample treatment was changed,the cells were pretreated by G2/M phase blocking drugs to re-infect the virus,the samples were collected,and the effects of G2/M phase blocking drugs on EV-D68 replication were further analyzed by Western Blot,RT-q PCR and TCID50/ml experimental methods.Results Compared with the control group(Infected + Con),the treatment groups of Emodin,Artemisinin and Astragaloside A(S phase arrest drugs)could not change the cycle arrest of EV-D68 manipulation;the intracellular viral m RNA levels were decreased respectively from 1.00 ± 0.03 to 0.78 ± 0.04(p<0.01),from 1.00 ± 0.14 to0.86 ± 0.11(p>0.05),from 1.00 ± 0.14 to 0.68 ± 0.11(p<0.05);TCID50/ml results showed no downward trend,and morphological observation did not show protection against host cells.Compared with the control group(Infected + Con),the percentage of cells in the G0/G1 phase of each treatment group of PAB,Oridonin and Erianin(G2/M phase blocker)decreased respectively from 49.18% ± 0.03% to 0.68% ± 0.54%(p<0.001),from 49.18% ± 0.63% to 38.04% ± 1.95%(p<0.001),and from 40.37% ± 1.34% to0.15% ± 0.25%(p<0.001).And the three drugs significantly changed the protein expression of Cyclin B1,Cdk1,P53,P21 and P27 regulated by EV-D68;morphological observation showed that they can protect the host cells and reduce the number of cell deaths.RT-q PCR results showed that the multiple difference difference between the control group(Infected + Con)and PAB,Oridonin and Erianin in the intracellular m RNA was respectively 2.33(p<0.001),6.67(p<0.001)and 2.56(p<0.001);TCID50/ml results showed that the multiple difference between TCID50/ml in the control group(Infected+Con)and PAB,Oridonin and Erianin was respectively 2.27× 102(p<0.01)? 5.12×103(p<0.01)and 1.23×104(p<0.01);Western Blot results showed that the multiple difference between VP1 protein expression in the control group(Infected + Con)and PAB,Oridonin and Erianin was respectively1.47(p<0.01)?1.33(p<0.01)and 2.22(p<0.01).The amount of virus in the cell supernatant was analyzed.RT-qPCR results showed that the multiple difference of the viral genome level in the supernatants of the control group(Infected + Con)and PAB,Oridonin and Erianin was respectively11.31(p<0.001)and 54.05(p<0.001)and 108.70(p<0.001);Western Blot results showed the fold difference between the expression of viral VP1 protein in the supernatant of the control group(Infected + Con)and PAB,Oridonin and Erianin was respectively 1.39(p<0.01),3.45(p<0.01)and 1.54(p<0.01).The results of the virus infection test after drug pretreatment were analyzed.RT-q PCR results showed that the difference of m RNA in the control group(Infected+ Con)and PAB,Oridonin and Erianin was respectively 2.66(p<0.001)? 4.68(p<0.001)and 2.5(p<0.001);Western Blot results showed that the multiple difference of VP1 expression in the control group(Infected + Con)and PAB,Oridonin and Erianin was respectively 9.09(p<0.01)?6.67(p<0.01)and 2.5(p< 0.01).At the same time,the difference in expression of VP1 in the supernatant was respectively 1.85(p<0.01),14.28(p<0.01),and 2.22(p<0.01).TCID50/ml results showed the multiple difference of the virus in the control group(Infected + Con)and PAB?Oridonin and Erianin was respectively 2.07×102(p<0.01),4.14×103(p<0.001)and 1.66 × 104(p <0.001).Conclusions1.Drugs that induce S-phase arrest cannot change the cell cycle state suitable for EV-D68 replication.Although it can slightly inhibit the m RNA level of the virus,it does not inhibit the level of viral virulence and can not protect the host cells.2.Drugs that induce G2/M phase arrest cell cycle status by altering the expression of Cyclin B1,Cdk1,P53,P21 and P27 proteins manipulated by EV-D68,decreasing the m RNA level of EV-D68 and the expression of VP1 protein and significantly reduce the number of progeny virus,protect the host cells,inhibit the replication and production of EV-D68.3.The key to the inhibition of viral replication by the drugs with G2/M phase arrest is not only in the process of viral genome replication and protein expression,but the key is the final assembly of the virus into the viral particle stage.4.The G2/M phase block drug has no inhibitory effect on the viral entry. |
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