Effect of total flavonoids of Spatholobus suberectus Dunn on PCV2 induced oxidative stress in RAW264.7 cells

Wan

et al. 2020

Preprint

Background: Porcine circovirus type 2 (PCV2) is an important DNA pathogen commonly in pig farms, which can cause immunosuppression and induce apoptosis. To evade clearance by the host immune system, PCV2 constantly builds up complex mechanisms or mutates genes, it is difficult to eradicate complex PCV2 infection by relying on vaccine immunity and one compound. At present, there is no literature report on the effective prevention and treatment of PCV2 infection by the combined a of two or more compounds through multiple pathways and multiple targets. We have previously demonstrated the anti-PCV2 effect of Matrine in vitro, but its mechanism has not been further studied. Many literatures have proved that Osthole has a variety of pharmacological activities. Therefore, this study explored the synergistic antiviral effect of Matrine combined with Osthole and the synergistic anti-apoptosis mechanism in the same pathway and targets.Results: It was first demonstrated that Osthole alone had anti-PCV2 effect and the synergistic anti-PCV2 effect combined with Matrine was better than Matrine and Osthole alone by qPCR, IFA and Western blot results. Subsequently, demonstrated that the reduction of endoplasmic reticulum PERK apoptosis induced by PCV2 was mainly contributed the mechanism of anti-PCV2 using these two extracts by Annexin V-FITC/PI, JC-1 and Western blot results. Finally, it was further demonstrated that Matrine combined with Osthole could inhibit the expression of Cap in cap-transfected PK-15 cells, thus inhibiting Cap-induced PERK apoptosis. In this study, Ribavirin was used as the positive control.Conclusions: It was proved that the synergistic anti-PCV2 effect of Matrine combined with Osthole was significantly better than that of Matrine and Osthole alone. And the two ombined, with Cap and GRP78 as potential targets of anti-PCV2, further inhibited PERK apoptosis induced by PCV2 infection by directly inhibiting the expression of PCV2 Cap protein. The results formed a basis for further studies on the mechanism of anti-PCV2 in vivo using Matrine combined with Osthole and developing new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets.

“…PCV2 has been reported to induce mitochondrial apoptosis in PK-15 cells [27,28]. MMP is recognized as a marker of mitochondrial statement.…”

Section: Discussionmentioning

confidence: 99%

The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2

Wan

et al. 2020

Preprint

“…Medical research shows that curcumin has low toxicity, little adverse reactions and multiple therapeutic effects, such as anti-inflammatory [29], anti-oxidative [30], anti-H9N2 influenza virus [31], dengue virus [32], hepatitis C virus [33] and so on. Zhang et al demonstrated that PCV2 Cap could induce mitochondrial apoptosis of PK-15 and PAM cells by interfering with Ca 2+ homeostasis, down-regulating MMP and increasing intracellular ROS [7]; Lin et al found that PCV2 induced mitochondrial apoptosis of PCV2 infected PK-15 cells and 293 T cells by increasing the activities of caspase-3 and caspase-9 and accelerating the release of cytochrome C (Cyto C) from mitochondria to cytoplasm [34]; After PCV2 infected RAW264.7 cells, stress-related molecules such as ROS production and NO secretion were significantly increased [35]; Previous studies have shown that PCV2 infection promotes the accumulation of ROS, which in turn inhibits the replication of PCV2 through the NF-κB pathway [36]. Therefore, we hypothesized that the experimental compounds could inhibit the mitochondrial apoptosis of PK-15 cells induced by PCV2 infection via acting on mitochondrial membrane potential.…”

Section: Discussionmentioning

confidence: 99%

Cepharanthine and Curcumin inhibited mitochondrial apoptosis induced by PCV2

Zheng

et al. 2020

BMC Vet Res

Background Porcine circovirus type 2 (PCV2) is an immunosuppressive pathogen with high prevalence rate in pig farms. It has caused serious economic losses to the global pig industry. Due to the rapid mutation of PCV2 strain and co-infection of different genotypes, vaccination could not eradicate the infection of PCV2. It is necessary to screen and develop effective new compounds and explore their anti-apoptotic mechanism. The 13 natural compounds were purchased, with a clear plant origin, chemical structure and content and specific biological activities. Results The maximum no-cytotoxic concentration (MNTC) and 50% cytotoxic concentration (CC50) of 13 tested compounds were obtained by the cytopathologic effect (CPE) assay and (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in PK-15 cells. The results of qPCR and Western blot showed that, compared with the PCV2 infected group, the expression of Cap in Paeonol (0.4 mg/mL and 0.2 mg/mL), Cepharanthine (0.003 mg/mL, 0.0015 mg/mL and 0.00075 mg/mL) and Curcumin (0.02 mg/mL, 0.001 mg/mL and 0.005 mg/mL) treated groups were significantly lowered in a dose-dependent manner. The results of Annexin V-FITC/PI, JC-1, Western blot and ROS analysis showed that the expression of cleaved caspase-3 and Bax were up-regulated Bcl-2 was down-regulated in Cepharanthine or Curcumin treated groups, while ROS and MMP value were decreased at different degrees and the apoptosis rate was reduced. In this study, Ribavirin was used as a positive control. Conclusions Paeonol, Cepharanthine and Curcumin have significant antiviral effect. And the PCV2-induced Mitochondrial apoptosis was mainly remitted by Cepharanthine and Curcumin.

“…Lin et al proved that the PCV2 could up-regulate MMP and induce apoptosis through mitochondrial pathway [34]; Chen et al proved the accumulation of ROS and interference with mitochondrial function were demonstrated in RAW264.7 cells infected with PCV2 [35,36]. Mitochondria have a central role in cell apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Cepharanthine and Curcumin inhibited mitochondrial apoptosis induced by PCV2

Zheng

et al. 2020

Preprint

Background Porcine circovirus type 2 (PCV2) is an immunosuppressive pathogen with high prevalence rate in pig farms. It has caused serious economic losses to the global pig industry. Due to the rapid variation of PCV2 strain, short vaccine protection period and poor cross-protection effect, etc. It is necessary to screen and develop effective new compounds and explore their anti-PCV2 mechanism. The 13 natural compounds were purchased, with a clear plant origin, chemical structure and content and specific biological activities. Methods The maximum no-cytotoxic concentration (MNTC) and 50% cytotoxic concentration (CC50) for 13 tested compounds were determined by the cytopathologic effect (CPE) assay and (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in PK-15 cells, respectively, qPCR and Western blot were used to detect the anti-PCV2 effect of compounds. Western blot method, AnnexinV-FITC /PI, JC-1, and ROS Kits were respectively used to detect the anti-PCV2 induced apoptosis mechanism of Cepharanthine and Curcumin. In this study, Ribavirin was used as the positive control. Results MNTC and CC50 of 13 compounds were obtained. Compared with the PCV2 infected group, the expression of Cap treated with Paeonol (0.4 mg/mL and 0.2 mg/mL), Cepharanthine (0.003 mg/mL, 0.0015 mg/mL and 0.00075 mg/mL) and Curcumin (0.02 mg/mL, 0.001 mg/mL and 0.005 mg/mL) were significantly lower in a dose-dependent manner. The expression of Cleaved-caspase3 and Bax were up-regulated in the treated with Cepharanthine or Curcumin, while the expression of Bcl-2 was down-regulated, ROS value and MMP value were decreased to different degrees and the apoptosis rate was reduced. Conclusion Paeonol, Cepharanthine and Curcumin have significant antiviral effect. And the PCV2-induced Mitochondrial apoptosis was mainly remitted by Cepharanthine and Curcumin.