Mechanistic Insights Of The Anti-Porcine Rotavirus Activity Of POL-P And Its Regulation Of The PI3K/AKT/mTOR Pathway

Porcine Rotavirus diarrhea is an acute viral gastroenteritis disease caused by porcine rotavirus（PoRV）,with diarrhea and dehydration as the main symptoms in piglets.The disease can lead to the death of piglets and cause significant economic losses to the pig breeding industry.Currently,PoRV vaccination is commonly used to prevent porcine rotavirus diarrhea,but its efficacy is not good.Therefore,it is of practical significance and urgency to find and develop safe and efficient new drugs against PoRV.Portulaca Oleracea L.（POL）has a sour taste,is non-toxic,has heat detoxification,invigorates the spleen,has antidiarrheal effects,and is beneficial for treating various diseases.POL is rich in nutrition and is recognized by the National Ministry of Health as a wild medicinal herb with the same source of medicine and food.It is also a high-quality feed resource for livestock and poultry,with high development and utilization value.Portulaca Oleracea L.Polysaccharide（POL-P）is an effective active ingredient of POL.Modern pharmacological studies have shown that POL-P has the effects of reducing toxins in the body,improving immunity,and treating ulcerative colitis.However,the effect of POL-P against PoRV infection and its molecular mechanism have not been reported.In order to further expand the clinical application of POL and considering the harm caused by PoRV and the detoxification and anti-dysentery activity of POL,the anti-PoRV effect and related mechanism of POL-P were investigated.This would provide a new theoretical basis for further improving the mechanism of PoRV infection and developing new drugs for the prevention and control of rotavirus.The research will focus on the following five aspects:1.Extraction,Isolation,and Identification of POL-P.To investigate the anti-PoRV infection effects of POL-P,this section of the experiment focuses on the extraction,isolation,and identification of POL-P.The ultrasonic method is used for the extraction of POL-P,and various analyses are conducted to determine the extraction conditions,polysaccharide content,relative molecular weight,and monosaccharide composition.The results reveal that the extraction yield of POL-P is determined to be 8.56%.The infrared results indicate that POL-P is a polysaccharide containingαandβglycosidic linkages.Gel column chromatography reveals a relative molecular weight of 4×10⁴ Da for POL-P.Monosaccharide composition analysis demonstrates that POL-P is composed of mannose,xylose,glucuronic acid,galacturonic acid,glucose,galactose,and arabinose,with relative masses of 1.2%,13.2%,33.5%,1.2%,3.3%,32.2%,and 5.4%,respectively.2.Research on the antiviral effects of POL-P against PoRV infection.After successfully extracting POL-P,the study focuses on investigating the anti-PoRV infection effects of POL-P.In this section of the experiment,the following techniques will be employed to assess the impact of POL-P on different stages of PoRV viral replication:real-time quantitative PCR,Western blot,TCID₅₀ assay,and indirect immunofluorescence assay.These techniques will be used to evaluate the levels of PoRV viral replication in vitro after treatment with POL-P.Additionally,ELISA will be utilized to measure the levels of inflammatory cytokines,clarifying the anti-PoRV infection effects of POL-P in vitro.Furthermore,an in vivo experiment will be conducted using PoRV-infected mice to further investigate the anti-PoRV infection effects of POL-P.Clinical condition scoring,clinical post-mortem examination,histopathological observations of tissues,and viral load measurements will be performed to determine the efficacy of POL-P in combating PoRV infection in vivo The results demonstrate that the optimal dosage of POL-P at 400μg/m L significantly reduces the expression levels of PoRV VP6 protein,nucleic acid,and viral particles during the internalization,replication,and entire process stages of PoRV infection.POL-P significantly reduces the expression levels of TNF-α,IL-6,and IL-10inflammatory cytokines during the internalization and replication process of PoRV infection.Additionally,POL-P significantly increases the expression of IFN-αcompared to the PoRV group.In vivo animal experimental results demonstrate that POL-P at a dose of 400.0 mg/kg exhibits therapeutic effects against PoRV infection in mice.3.Network pharmacology of PoRV treated with POL-P.According to the experimental results,a total of 296 effective targets were predicted for the signaling pathway of POL-P against PoRV.These targets are likely associated with the antiviral activity of POL-P,and further analysis can reveal the mechanism of action and regulatory pathways of POL-P against PoRV.POL-P and PoRV share 16 intersecting targets,mainly enriched in the PI3K/AKT signaling pathway.These results provide a theoretical basis for further elucidating the mechanism of action of POL-P against PoRV.4.Study on the antiviral effects of POL-P against PoRV through the inhibition of the PI3K/AKT/mTOR pathway.To validate the mechanism of action of POL-P against PoRV through regulation of the PI3K/AKT/mTOR pathway,this chapter’s experiment employed Western blot and TCID₅₀ methods to determine the impact of POL-P and PoRV on the PI3K/AKT/mTOR signaling pathway during the virus internalization and replication stages.Based on the results confirming POL-P’s inhibition of virus internalization and replication stages,pathway protein inhibitors and activators were used to intervene,elucidating the mechanism of POL-P’s anti-PoRV activity through the PI3K/AKT/mTOR signaling pathway.These experiments will help uncover the mode of action of POL-P and validate its ability to combat PoRV infection by modulating the PI3K/AKT/mTOR signaling pathway.During PoRV infection,POL-P significantly inhibits PoRV by modulating the PI3K/AKT/mTOR signaling pathway.It suppresses the expression of p-PI3K during the internalization process and inhibits the expression of p-AKT during the replication process,leading to a significant inhibition of PoRV.5.Effects of Portulaca oleracea Polysaccharide（POL-P）on Porcine Rotavirus Infection and Intestinal Microbiota in Piglets.To evaluate the effects of POL-P on piglets infected with PoRV,an in vivo animal experiment was conducted.Clinical condition scoring,clinical autopsy,histopathology,blood parameters,and ELISA were performed to evaluate the therapeutic effect of POL-P on PoRV-infected piglets.Furthermore,16S rRNA sequencing of piglet fecal samples was conducted using the Illumina Nova Seq 6000 platform to analyze the impact of POL-P on the intestinal microbiota.The results showed that POL-P at a dose of 50 mg/kg shows therapeutic effects against PoRV infection in piglets.Blood routine and biochemical parameters in the PoRV+POL-P group returned to normal levels,indicating significant improvement in piglet dehydration compared to the PoRV group.The viral load in the jejunum,ileum,and feces of the PoRV+POL-P group was significantly lower than that in the PoRV group.Compared to the PoRV group,POL-P can alter the structure of piglet gut microbiota,further improving the dysbiosis caused by PoRV and providing protection against piglet diarrhea.These findings suggest that POL-P exhibits therapeutic effects on PoRV-induced diarrhea and restoration of the intestinal microbiota.In summary,POL-P can inhibit PoRV replication in vitro and in vivo pathogenicity,and further inhibit PoRV replication by inhibiting the activation of the PI3K/AKT/mTOR signaling pathway.This experiment provides new insights into the infection mechanism of PoRV and provides new ideas for virus prevention and control.