Preliminary Study On The Effect And Mechanism Of Qilian Powder On PRRS Vaccine Immunity

Porcine reproductive and respiratory syndrome(PRRS)is a viral infection caused by PRRSV(Porcine reproductive and respiratory syndrome virus,PRRSV)that seriously affects the swine farming industry.PRRS can lead to reproductive disorders in boars,fever and abortion in gestating sows and increased mortality in newborn piglets,causing huge economic losses to the swine farming industry.Current treatments for PRRS are limited and rely heavily on the use of antibiotics and antivirals,and vaccines are usually used for prevention.However,the immune escape characteristic of PRRSV infection makes traditional vaccines have limited effect on its immunity.In contrast,traditional Chinese medicine has shown good effects in regulating immune function in recent years,with the function of activating and enhancing the body’s immune system to play a role in improving the immune effect of vaccines.Qilian Powder(QLP),is composed of Astragali Radix,Forsythiae Fructus and bitter ginseng.In this formula,Astragali Radix tonifies Qi,promotes Yang,generates fluid and nourishes blood;it is the main herb;Forsythiae Fructus clears heat and detoxifies the body,cools the surface and is a supplementary herb;Sophorae Flavescentis Radix clears heat and dampness and is an adjuvant;all the herbs together work to tonify Qi and nourish Blood,clear heat and dry dampness.In this study,we investigated the effect of QLP on the immune function of piglets after immunization with PRRSV,and verified the mechanism of QLP and its main active ingredients in regulating immunity,so as to provide a theoretical basis for the clinical development of safe and effective immune enhancers.The optimal ratio of QLP was firstly screened by orthogonal test design,and the optimal ratio of Astragalus membranaceus was obtained as 2:1:1 by testing the PRRSV load and IFN-β.Subsequently,clinical validation was carried out according to the screened optimal ratios.Thirty weaned piglets were acclimatized and reared for 7 days,and then randomly divided into 5 groups: group A,PRRSV immunized group;group B,high dose group of QLP(0.5 g/kg);group C,medium dose group of QLP(0.25 g/kg);group D,low dose group of QLP(0.125 g/kg group);and group E,the group of positive medication(Fuzhengjiedu powder).Each dosing group was administered continuously for 2 weeks,followed by 2 weeks of observation.The PRRSV antibody level,immunoglobulin level,T-lymphocyte subpopulation changes,immune cytokine level,peripheral blood lymphocyte proliferation rate,erythrocyte immune function,oxidative stress level,physiological and biochemical indexes,and growth performance were comprehensively assessed from 1 week of administration to 2 weeks of drug cessation with a view to obtaining the clinically recommended dosage for use.The results showed that QLP could significantly increase the levels of PRRSV antibody,immunoglobulin and T lymphocyte subpopulation,and thus play a role in improving the adaptive immune function of PRRSV-immunized pigs.QLP can significantly increase the secretion of IL-2,IL-4,GM-CSF and IFN-β,and inhibit the secretion of IL-10 in PRRSV-immunized pigs.QLP can improve the innate immune function of PRRSVimmunized pigs by enhancing the proliferation rate of peripheral blood lymphocytes and erythrocyte immunity.The results of blood physiological and biochemical indexes showed that QLP could increase the number of innate immune cells and improve the utilization rate of feed nutrients in the immunized pigs,so as to improve the growth performance of piglets.Taking into account all the test results and the cost of the drug,the middle dose of QLP(0.25g/kg)was finally selected as the recommended clinical dose.Finally,the modulatory effect of Astragaloside IV(AS-IV),the main active ingredient of astragaloside,on the c GAS-STING pathway was verified.The MTT results showed that AS-IV was not cytotoxic to PAMs in the concentration range of 0-200 μg/m L.50 μg/m L,25 μg/m L,and 12.5 μg/m L were selected for the subsequent experiments.All doses of AS-IV significantly inhibited the proliferation of PRRSV in PAMs and significantly increased the secretion of IFN-β from PAMs.The innate immune function of PAMs was examined by ELISA and RT-q PCR,and the results showed that AS-IV significantly increased the secretion and m RNA expression of the antigen-presenting molecules SLA-Ι,SLA-II,CD86,and CD80,and significantly increased the secretion and m RNA expression of the innate immune function molecules,Nramp and SP-A,in PAMs.Subsequently,AS-IV regulation of the c GAS-STING pathway was validated,and the results showed that AS-IV significantly ameliorated the inhibition of PRRSV infection leading to the suppression of c GAS and TBK1 m RNA and protein expression and inhibited the activation of IRF-3 and STING by PRRSV infection.This was followed by the testing of IFN-β and ISGs,and it was found that PRRSV infection would inhibit m RNA expression of IFN-β,OAS1 and ISG15,while AS-IV significantly attenuated their inhibition.In addition,c GAMP,a product of the c GAS-STING signaling pathway,was detected using ELISA,and the results showed that the secretion of c GAMP was significantly reduced by PRRSV infection,and ASIV significantly restored its secretion level.c GAS-STING pathway also mediates inflammation,and AS-IV significantly inhibited the inflammatory response induced by PRRSV infection,as evidenced by the inhibiting the expression of inflammatory cytokines IL-1β,IL-6 and TNF-α and the expression of NF-κB.The above results suggest that AS-IV can inhibit the inflammatory response induced by PRRSV infection by regulating the c GAS-STING signaling pathway AS-IV significantly attenuated the inhibitory effect of c GAS-STING signaling pathway caused by PRRSV infection,restored the expression of IFN-β and interferon-stimulated genes,and inhibited the inflammatory response induced by PRRSV infection.In conclusion,the above findings showed that QLP showed good effects in regulating the immune function of PRRSV,providing a scientific basis for promoting the application and promotion of Chinese herbal compound preparations in pig production,as well as providing a safer and more environmentally friendly method for the sustainable development of livestock and poultry farming,and promoting the healthy development of the farming industry.