Hongquan Li

Effect of selenylation modification on immune-enhancing activity of Atractylodes macrocephala polysaccharide

The Atractylodes macrocephala polysaccharide (AMP) was extracted purified and modified in selenylation by Nitric acid-sodium selenite method to get nine selenizing AMPs (sAMPs), sAMP(1)-sAMP(9). In vitro test their effects on chicken peripheral lymphocyte proliferation were determined by MTT assay. The results showed that nine sAMPs and AMP at five concentrations could significantly promote lymphocyte proliferation, the actions in six sAMPs were significantly stronger than that in AMP, and in sAMP(9) was the strongest. In vivo test, 14-day-old chickens vaccinated with ND vaccine were injected respectively with sAMP(9) and AMP, the peripheral lymphocytes proliferation, serum antibody titer, IFN-γ, IL-2 and IL-6 contents were determined. The results displayed that the sAMP could significantly promote lymphocyte proliferation and elevate the antibody titers and content of IFN-γ, IL-2 and IL-6 in comparison with unmodified AMP. These results indicate that selenylation modification can significantly enhance the immune-enhancing activity of AMP.

Optimization of selenylation modification for garlic polysaccharide based on immune-enhancing activity.

Garlic polysaccharide (GPS) was modified in selenylation respectively by nitric acid-sodium selenite (NA-SS), glacial acetic acid-selenous acid (GA-SA), glacial acetic acid-sodium selenite (GA-SS) and selenium oxychloride (SOC) methods each under nine modification conditions of L9(3(4)) orthogonal design and each to obtain nine selenizing GPSs (sGPSs). Their structures were identified, yields and selenium contents were determined, selenium yields were calculated, and the immune-enhancing activities of four sGPSs with higher selenium yields were compared taking unmodified GPS as control. The results showed that among four methods the selenylation efficiency of NA-SS method were the highest, the activity of sGPS5 was the strongest and significantly stronger than that of unmodified GPS. This indicates that selenylation modification can significantly enhance the immune-enhancing activity of GPS, NA-SS method is the best method and the optimal conditions are 0.8:1 weight ratio of sodium selenite to GPS, reaction temperature of 70 °C and reaction time of 10h.

The comparison of antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide (CP) and sulfated CP

Codonopsis pilosula polysaccharide (CP) was extracted, purified and modified by chlorosulfonic acid-pyridine method to obtain a sulfated CP (sCP). Their antioxidative activities in vitro were compared through the free radical-scavenging test. The results demonstrated that the scavenging capabilities of sCP were significantly stronger than those of CP. In vivo test, the mice hepatic injury model was prepared by BCG/LPS method, then administrated respectively with sCP and CP at three dosages, the biochemical indexes in serum, antioxidative indexes in liver homogenate and histopathological change in liver of the mice were compared. The results showed that in high (200mg/kg) and middle (150mg/kg) dosages of sCP groups, the contents of ALT, AST and TNF-α in serum and MDA in liver homogenate were significantly lower than those in the model group and numerically lower than those in the CP groups, the activities of SOD and GSH-Px in liver homogenate were significantly higher than those in the model group and numerically higher than those in the CP groups. In the model group there were obvious pathological changes in the liver, while in the sCP groups were near normal. These results indicate that sCP and CP possess antioxidative activity in vitro and in vivo, the activity of sCP is stronger than that of CP and sulfation modification can enhance the antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide.

Anti-PRRSV effect and mechanism of sodium tanshinone IIA sulfonate in vitro

This experiment was conducted to study the antiviral activities of sodium tanshinone IIA sulfonate (STS) against porcine reproductive and respiratory syndrome virus (PRRSV) and its mechanism. Anti-PRRSV activities of STS were observed on Marc-145 cells by using visualization of cytopathologic effect assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, and its antiviral mechanism was determined by time-of-addition assay, adsorption inhibition assay, and virucidal assay. The results showed that STS could reduce the damage of PRRSV to Marc-145 cells, with the inhibition ratio exceeding to 100%, at the maximum non-cytotoxic concentration. The time-of-addition and virucidal assays indicated that the anti-PRRSV activities of STS could be due to inhibiting the virus replication or/and inactivating the virus directly. The inhibition of the virus attachment was not discovered in adsorption inhibition assay. The results proved that STS had strong anti-PRRSV activity and encouraged for further exploration of STS.

Modification of lily polysaccharide by selenylation and the immune-enhancing activity.

Lily polysaccharide (LP) was extracted, purified and selenizingly modified by HNO3-Na2SeO3 method according to L9(3(4)) orthogonal design. Nine selenizing LPs, sLP1-sLP9, were obtained and their immune-enhancing activities were compared taking unmodified LP as control. The results in vitro test showed that sLP6 presented the strongest activity in promoting lymphocytes proliferation in single and synergetic with PHA, and the relative expression level of IL-2, IL-6 and IFN-γ mRNA of chicken peripheral lymphocytes. The results in vivo test showed that sLP6 could promote lymphocytes proliferation and enhance the serum antibody titers and serum IL-2, IL-6, IFN-γ contents more significantly than LP in chickens vaccinated with Newcastle Disease (ND) vaccine. These results indicate that polysaccharide selenizing can significantly enhance the immune-enhancing activity of LP and the optimal modification conditions are 400 mg of Na2SeO3 per 500 mg of LP, the reaction temperature of 70 °C and the reaction time of 6 h.

Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

The co-infection of porcine reproductive respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) is quite common in clinical settings and no effective treatment to the co-infection is available. In this study, we established the porcine alveolar macrophages (PAM) cells model co-infected with PRRSV/PCV2 with modification in vitro, and investigated the antiviral activity of Matrine on this cell model and further evaluated the effect of Matrine on virus-induced TLR3,4/NF-κB/TNF-α pathway. The results demonstrated PAM cells inoculated with PRRSV followed by PCV2 2 h later enhanced PRRSV and PCV2 replications. Matrine treatment suppressed both PRRSV and PCV2 infection at 12 h post infection. Furthermore, PRRSV/PCV2 co- infection induced IκBα degradation and phosphorylation as well as the translocation of NF-κB from the cytoplasm to the nucleus indicating that PRRSV/PCV2 co-infection induced NF-κB activation. Matrine treatment significantly down-regulated the expression of TLR3, TLR4 and TNF-α although it, to some extent, suppressed p-IκBα expression, suggesting that TLR3,4/NF-κB/TNF-α pathway play an important role of Matrine in combating PRRSV/PCV2 co-infection. It is concluded that Matrine possesses activity against PRRSV/PCV2 co-infection in vitro and suppression of the TLR3,4/NF-κB/TNF-α pathway as an important underlying molecular mechanism. These findings warrant Matrine to be further explored for its antiviral activity in clinical settings.

Antiviral activity and underlying molecular mechanisms of Matrine against porcine reproductive and respiratory syndrome virus in vitro

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is an acute infectious disease. The prevalence of PRRS has made swine industry suffered huge financial losses. Matrine, a natural compound, has been demonstrated to possess anti-PRRSV activity in Marc-145 cells. However, the underlying molecular mechanisms were still unknown. The main objective of our study was to discuss the effect of Matrine on PRRSV N protein expression and PRRSV induced apoptosis. Indirect immunofluorescence assay (IFA) and Western blot were used to assess the effect of Matrine on N protein expression. Apoptosis was analyzed by fluorescence staining. In addition, the effect of Matrine on caspase-3 activation was investigated by Western blot. Indirect immunofluorescence assay and Western blot analysis demonstrated that Matrine could inhibit N protein expression in Marc-145 cells. And Matrine was found to be able to impair PRRSV-induced apoptosis by inhibiting caspase-3 activation.

In vitro antiviral activity and underlying molecular mechanisms of dipotassium glycyrrhetate against porcine reproductive and respiratory syndrome virus.

BACKGROUND Porcine reproductive and respiratory syndrome virus (PRRSV) has caused large economic losses in the swine industry. Currently, there is no effective way to prevent PRRSV infection. In this study, we investigated the inhibitory effect of dipotassium glycyrrhetate (DG), a derivative of glycyrrhetinic acid, on PRRSV infection ability. METHODS The cytotoxicity of DG was measured by MTT assay, and the effects of DG on PRRSV N gene/protein were investigated using real-time PCR, western blot and immunofluorescence assay. In addition, the effect of DG on cell apoptosis was analysed by fluorescence staining. RESULTS Our results indicated that DG could effectively inhibit virus replication and N gene expression in MARC-145 cells infected with PRRSV. When the infected cells received DG, the numbers of apoptotic cells were decreased, and the cleaved caspase-3 contents were decreased dramatically. CONCLUSIONS Our study demonstrates that DG could effectively inhibit the PRRS virus via multiple pathways including inhibition of virus replication and N gene expression and reduction of apoptotic cells. DG can serve as a potential chemical for PRRSV prevention and control.

Sodium tanshinone IIA sulfonate inhibits porcine reproductive and respiratory syndrome virus via suppressing N gene expression and blocking virus-induced apoptosis.

BACKGROUND Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic loss in the swine industry. Currently, there is no effective way to prevent PRRSV infection. Sodium tanshinone IIA sulfonate (STS), a natural compound derived from Salvia miltiorrhiza, was shown to possess anti-PRRSV activity, but the underlying mechanisms remain unclear. The objective of this study was to investigate the effect of STS on PRRSV-induced cell apoptosis and PRRSV N protein expression pattern. METHODS Relative quantification real-time PCR was used to evaluate the inhibition of STS on N gene expression. Simultaneously indirect immunofluorescence assay (IFA) and western blot were used to assess the effect on N protein expression. Apoptosis was analysed using fluorescence microscope with an annexin V-EGFP kit. The effect of STS on caspase-3 cleaving was assessed by western blot. RESULTS Our results showed that STS could inhibit viral N gene expression at both the messenger RNA stage and at the protein level in PRRSV-infected cells in a dose-dependent manner. In addition, STS could also rescue PRRSV-induced apoptosis. CONCLUSIONS Our data suggest that STS may serve as a base compound for developing more effective drugs against PRRSV infection.

In vitro evaluation of antiviral activity of tea seed saponins against porcine reproductive and respiratory syndrome virus.

BACKGROUND Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major swine pathogens. This virus causes immune suppression and other secondary infections, leading to significant economic losses in the swine industry. Tea seed saponins (TS) are a natural extract from tea seeds with anti-cancer, anti-inflammatory and antiviral activity. In this study, we demonstrated that TS possessed anti-PRRSV activity. METHODS MTT assay and trypan blue staining were used to evaluate the cytotoxicity and antiviral ability of TS in cell culture. Apoptosis was measured to assess the safety of TS on Marc-145 cells. Time-of-addition assay, entry inhibition assay and virucidal assay were used to assess the antiviral action of TS. The effect of TS on host cellular gene expression was analysed by real-time PCR. Absolute quantification RT-PCR and western blot were used to study the inhibitory effect of TS on PRRSV N gene and protein expression. RESULTS Our results showed that 50% cytotoxic concentrations (CC50) and 50% effective concentration (EC50) of TS were 59.86 ±0.3841 µg/ml and 24.29 ±1.194 μg/ml, respectively. The maximum non-cytotoxic concentration of TS on Marc-145 cells was 30 μg/ml. TS inhibited PRRSV-induced cell apoptosis and effectively inhibited PRRSV replication by reducing the expression of host cellular gene PABP, and significantly inhibited virus N gene/protein expression. CONCLUSIONS TS possessed anti-PRRSV activity in vitro and could serve as a potential antiviral drug for PRRSV prevention and control.