| Porcine circovirus type 2(PCV2)is an important immunosuppressive pathogen in pigs,which has induced apoptosis in vivo and in vitro.Vaccination is currently the main means of preventing the disease.However,PCV2 strain mutated rapidly,the vaccine protection period was short and the cross-protection effect of existing vaccines was poor.PCV2 belongs to immunosuppressive viruses.In co-evolution with the host,in order to escape the recognition and elimination of the host immune system,PCV2 continuously establishes a complex mechanism to survive.it is difficult to effectively control the complex PCV2 infection with a single drug.In the early stage,this research group has proved that Matrine has a significant anti-PCV2 effect.Many literatures have proved that Osthole is widely used in the medical field and has remarkable clinical efficacy.It has pharmacological activities such as anti-viral,anti-inflammatory,anti-cancer,anti-tumor and immune function enhancement.However,it is difficult to effectively control complex viral diseases with single prescription.Based on the traditional Chinese medicine compound as the theoretical basis,the active ingredients of traditional Chinese medicine are designed in a reasonable way to form a prescription with clear drug composition and clear mechanism of action.It is beneficial for the treatment of diseases with multiple pathways and targets.At present,there is no literature report on the prevention and control of PCV2 with the combination of Chinese medicine active ingredients.In the experiment,the clinical cure rate and total efficiency of Sophocarpidine(Matrine is its main active ingredient)combined with Fructus cnidii(Osthole is its main active ingredient)retrieved from the Chiese and English detaabase were analyzed using Rev Man 5.3.Matrine and Osthole were the source of the prescription and the anti-PCV2 effect and anti-apoptosis mechanism of Matrine combined with Osthole were investigated in vivo and in vitro by establishing the model of PCV2(Cap)infection PK-15 cell and the model of PCV2 infection kunming mouse.The results of in vitro experiments showed that: 1.The cytotoxicity of Osthole,Matrine combined with Osthole and Ribavirin on PK-15 cells were detected by CPE and CCK8 methods.The results showed that the maximum safe concentrations(MNTC)of Osthole,Matrine combined with Osthole and Ribavirin on PK-15 cells were 0.01 mg/mL,0.5 mg/mL +0.01 mg/mL and 0.5 mg/mL,respectively.2.The expression levels of PCV2 Cap gene and Cap protein in PCV2 infected cells after 48 h of compound treatment were detected by q PCR,IFA and Western blot.The results showed that Compared with the PCV2 infected group,Matrine combined with Osthole could significantly reduce the expression of Cap gene and Cap protein and there was dose dependence(P<0.05)and showed dose dependence.Compared with 0.5 mg/mL Matrine and 0.01mg/mL Osthole alone,Matrine combined with Osthole at high,medium and low doses significantly reduced the expression of Cap gene or protein at(P<0.05).Compared with the 0.5 mg/mL Ribavirin positive control group,Matrine combined with Osthole significantly reduced the expression of Cap gene or protein at 0.5 mg/mL+0.01mg/mL(P<0.05).The results showed that Matrine combined with Osthole had anti-PCV2 effect in vitro.3.The results of Annexin V-FITC/PI showed that the apoptosis rate of PCV2 infection group was significantly higher than that of the cell control group(P<0.05).Compared with the PCV2 infection group,the apoptosis rate of all the groups treated with Matrine combined with Osthole at high,medium and low doses significantly decreased(P<0.05).The results of JC-1 showed that compared with the control group,MMP in the PCV2 infection group was significantly increased(P<0.05).Compared with the PCV2 infection group,the expression of MMP was significantly decreased in the Matrine combined with Osthole at high,medium and low doses group(P<0.05).The results of Western blot showed that,compared with the control group,the expressions of GRP78,p-PERK?p-e IF2?,ATF4,CHOP,Cleaved caspase 3 and Bax proteins in the PCV2 infected group were significantly increased(P<0.05)and the expressions of the proteins of Bcl-2 were significantly decreased(P<0.05).Compared with the PCV2 infection group,the expression of pro-apoptotic proteins in the GRP78 and PERK pathway in the Matrine combined with Osthole at high,medium and low doses was significantly reduced(P<0.05)and the expression of Bcl-2 protein was significantly increased(P<0.05).Matrine combined with Osthole significantly reduced Cleaved caspase 9 expression at 0.5 mg/mL+0.01mg/mL(P<0.05).It showed that Matrine combined with Osthole targeted GRP78 and interfered with PCV2-induced apoptosis through the PERK pathway.4.Establish the model of cap-transfected PK-15 cells and induce apoptosis,and detect the changes of Cap proteins,GRP78 proteins and other apoptosis-related proteins in the PERK pathway by Western blot.The results showed that Compared with the control group,the expression of pro-apoptotic proteins of Cap,GRP78 and PERK pathways was significantly increased in the Cap transfection group(P<0.05),and the expression of Bcl-2 was significantly decreased(P<0.05).compared with the Cap infected group,the expression of Cap?GRP78?p-PERK?p-e IF2??ATF4?CHOP?Cleaved-caspase 3 and Bax in the Matrine combined with Osthole at high,medium and low doses was significantly reduced and the expression of Bcl-2 was increased(P<0.05).It was shown that Matrine combined with Osthole targets Cap and GRP78 and interferes with cap-induced apoptosis through the PERK pathway.The results of in vivo experiments showed that: 1.Cap genes in the tissues of mice infected with PCV2 for 5 days were detected by PCR and q PCR.The results of PCR showed that PCV2 Cap gene could be detected in liver,thymus,spleen,inguinal lymph node and lung of PCV2 infected mice.The results of q PCR showed that PCV2 was the most expressed in liver of mice(8.26×105 copies/ L),followed by thymus(2.28×105 copies/ L)and spleen(1.13×105 copies/ L).It could be seen that the mice model of PCV2 infection was successfully established and the mice infected with PCV2.It shows that the model of mouse has been successfully established.2.The expression of Cap gene in the livers of mice infected with PCV2 at 8 d(3 d after medication),11 d(6 d after medication)and 14 d(9 d after medication)were first detected by q PCR.The results showed that Matrine combined with Osthole(40 mg/mL+12 mg/mL)and(40 mg/mL+12 mg/mL)significantly reduced the expression level of Cap gene at 8,11 and 14 d in the liver with dose dependence(P<0.05).Compared with the PCV2 group infected with PCV2 on 8,11 and 14 d,the PCV2 group infected with 11 d had the highest viral load(6.14×108 copies/?L).The expression of Cap protein in the liver of PCV2 infected mice for 11d(6 d)was detected by Western blot.Compared with the PCV2 infected group,Matrine combined with Osthole at high,medium and low doses all significantly reduced the expression of Cap protein with dose dependence.Compared with treatment group of each compound,the Cap protein expression of Matrine combined with Osthole(40 mg/mL+12 mg/mL)was significantly lower than that of Matrine(40 mg/mL),Osthole(12 mg/mL)and Ribavirin positive control group(40 mg/mL)(P<0.05).The results showed that Matrine combined with Osthole had anti-PCV2 effect in vivo.3.The changes of Cleaved-caspase 3 in spleen of PCV2 infected mice at 11 d were detected by IHC.The results showed that Matrine combined with Osthole inhibited the expression of apoptin Cleaved-caspase 3.Western blot results showed that compared with the normal control group,the expression levels of GRP78,p-PERK,p-e IF2?,ATF4,CHOP,Cleaved-caspase 3 and Bax significantly decreased and the expression of Bcl-2 was increased(P<0.05).the expression of GRP78 and every proteins of PERK pathways in the PCV2 infection group was significantly increased(P<0.05)and the expression of Bcl-2 was significantly decreased(P<0.05).Compared with the PCV2 group,Matrine combined with Osthole significantly reduced the expression of GRP78,p-PERK,p-e IF2?,ATF4,CHOP,Cleaved-caspase 3 and Bax proteins at 40 mg/mL+12 mg/mL,20 mg/mL+6 mg/mL(P<0.05),and significantly increased the expression of bcl-2 proteins(P<0.05).Compared with the single Matrine,Osthole or Ribavirin groups,Matrine combined with Osthole at different concentrations significantly reduced the expression of GRP78(P<0.05).Matrine combined with Osthole significantly reduced the expression of p-PERK and CHOP at 40 mg/mL+12 mg/mL,20 mg/mL+6 mg/mL(P<0.05)and significantly increased the expression of Bcl-2 (P<0.05),while Matrine combined with Osthole(10 mg/mL+3 mg/mL)had no significant effect on the decrease of p-PERK,CHOP and increase of Bcl-2(P>0.05).It showed that Matrine combined with Osthole targeted GRP78 and interfered with pcv2-induced spleen cell apoptosis through the PERK pathway.Matrine combined with Osthole significantly reduced the expression of Cleaved-caspase 3 at 40 mg/mL+12 mg/mL(P<0.05),while Matrine combined with Osthole at 20 mg/mL+6 mg/mL,10 mg/mL+3 mg/mL had no significant effect on the expression of Cleaved-caspase 3(P>0.05).Matrine combined with Osthole at 40 mg/mL+12 mg/mL had no significant effect on the expression of p-PERK,ATF4 and Bax proteins(P>0.05).This study confirmed that Matrine combined with Osthole has synergistic anti-PCV2 effect and explained its mechanism of interfering with apoptosis through PERK pathway.It provides a theoretical basis for the follow-up research of Matrine combined with Osthole to develop new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets. |
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