Prophylactic Effect And Mechanism Of Tibetan Turnip Against Hypoxic Pulmonary/Cerebral Edema

Tibetan turnip(Brassica rapa ssp.Rapa,also known as "Yuan Gen"),which is of edible,feeding,and pharmaceutical values and adapted to the extreme environments of the Qinghai-Tibet Plateau,was used to relieve hypoxia and anti-hypoxia by Tibetan nationality traditionally.Previous studies of our research group revealed that n-butanol fraction of Tibetan Turnip(n-bu-TT)was its effective anti-hypoxia component,p-coumaric acid(CA)and p-coumaric acid-?-D-glucopyranoside(CAG)were main effective components.Further study proved that n-bu-TT and CA possess good prophylactic effect against hypoxic pulmonary edema and may also exert prophylactic effect against hypoxic cerebral edema.Based on previous studies,this study established acute nombaric hypoxic pulmonary/cerebral edema animal model and investigate the efficacy of n-bu-TT and CA against hypoxic pulmonary/cerebral edema.Then,we established physical-hypoxia cellular models of primary mice pulmonary microvascular endothelial cell and cerebral astrocyte cell to investigate the mechanism of CA against hypoxic pulmonary/cerebral edema.The main research contents and results were listed as below:1.Acute nombaric hypoxic pulmonary edema ICR mice model was established and the effective dose range of n-bu-TT and CA prophylactic against hypoxic pulmonary edema were studied.Then,100 healthy male ICR mice were randomly assigned to 5 groups,including one normoxia control group and four hypoxia treatment groups,20 mice in each group.The normoxia control group was gavaged with sterilized water,four hypoxia treatment groups were gavaged with sterilized water,400 mg/kg.bw n-bu-TT,100 mg/kg.bw CA and 2 mg/kg.bw dexamethasone(positive drug control),once daily for four consecutive days.One hour after the final gavage,mice in hypoxia treatment groups were put into normobaric hypoxia chamber(9.5 ± 0.2%O2)for 24 h.After the hypoxia exposure,lung water content(LWC),pulmonary vascular permeability,protein content of bronchoalveolar lavage fluid(BALF),plasma total nitric oxide(NO)and endothelin-1(ET-1)content were determined in each groups;histological and ultra-microstructure analyses were performed.Expression of tight junction protein Occludin in lung tissue was assayed by immunohistochemistry.The results revealed that under hypoxic environment of 9.5%O2,mice treated with 400 mg/kg.bw n-bu-TT and 100 mg/kg.bw CA had significantly lower LWC and BALF protein content than hypoxia vehicle group mice.Meanwhile,both above groups of mice showed intact lung blood-gas-barrier,increased plasma total NO level,decreased plasma ET-1 level and upregulation of Occludin.The preventive effect of n-bu-TT and CA against hypoxic pulmonary edema was similar to the commonly used drug(dexamethasone).2.Acute nombaric hypoxic cerebral edema ICR mice model was established and the effective dose range of n-bu-TT and CA prophylactic against hypoxic cerebral edema were studied.Then,100 healthy male ICR mice were randomly assigned to 5 groups,including one normoxia control group and four hypoxia treatment groups,20 mice in each group.The normoxia control group was gavaged with sterilized water,four hypoxia treatment groups were gavaged with sterilized water,400 mg/kg.bw n-bu-TT,100 mg/kg.bw C A and 2 mg/kg.bw dexamethasone,once daily for four consecutive days.One hour after the final gavage,mice in hypoxia treatment groups were put into normobaric hypoxia chamber(9.5 ± 0.2%O2)for 24 h.After the hypoxia exposure,brain water content(BWC),blood-brain barrier permeability,Na+-K+-ATPase activity,mitochondrial membrane potential,oxidative stress and inflammatory cytokines were determined in each groups;histological and ultra-microstructure analyses were performed.Expression of tight junction protein Occludin in cerebral cortex was assayed by immunohistochemistry.The results revealed that under hypoxic environment of 9.5%O2,mice treated with 400 mg/kg.bw n-bu-TT and 100 mg/kg.bw CA had much lower BWC and blood-brain barrier permeability than hypoxia vehicle group mice.Meanwhile,both above two groups of mice showed increased Na+-K+-ATPase activity,mitochondrial membrane potential,decreased oxidative stress and inflammatory cytokines,and upregulation of Occludin.The preventive effect of n-bu-TT and CA against hypoxic cerebral edema was similar to dexamethasone.3.Physical-hypoxia models of primary mice pulmonary microvascular endothelial cell was established to investigate the mechanism of CA against hypoxic pulmonary edema.Then 5 groups were set up,including normoxia control group,hypoxia vehicle group,hypoxia+25 ?mol/L CA group,hypoxia+50 ?mol/L CA group and hypoxia+10?mol/L dexamethasone group.After pretreatment in normoxia condition for 1 h,four hypoxia treatment groups were put into a tri-gas incubator(1%O2+5%CO2+94%N2)for 24 h.Cell viability,NO and ET-1 released in cell culture supernant were determined.The mRNA level of HIF-1?,VEGF,iNOS,eNOS and ET-1 was assayed and ultra-microstructure analyses of primary pulmonary microvascular endothelial cell in each group were performed.Then,RT-qPCR and Western Blot were utilized to determine the mRNA and protein level of HIF-1?,VEGF,iNOS,eNOS and ET-1 in lung tissues of hypoxic pulmonary edema mice model.Results showed that the molecular mechanism of CA against hypoxic pulmonary edema was associated with reduction of mRNA and protein level of HIF-1?,VEGF,iNOS,eNOS and ET-1,which would lead to reduction of pulmonary vascular permeability,incresement of NO release and decrement of ET-1 synthesis.4.Physical-hypoxia models of primary mice cerebral astrocyte cell was established to investigate the mechanism of CA against hypoxic cerebral edema.Then 5 groups were set up,including normoxia control group,hypoxia vehicle group,hypoxia+25 ?gmol/L CA group,hypoxia+50 ?mol/L CA group and hypoxia+25 ?mol/L dexamethasone group.After pretreatment in normoxia condition for 1 h,four hypoxia treatment groups were put into a tri-gas incubator(1%O2+5%CO2+94%N2)for 24 h.Cell viability,the mRNA level of HIF-1?,VEGF,ET-1 and AQP4 were assayed and ultra-microstructure analyses of primary cerebral astrocyte cell in each group were performed.Then,RT-qPCR and Western Blot were utilized to determine the mRNA and protein level of HIF-1?,VEGF,ET-1 and AQP4 in cerebral cortex of hypoxic cerebral edema mice model.Results showed that the major efficacy mechanism of CA against hypoxic cerebral edema was associated with reduction of mRNA and protein level of HIF-1?,VEGF,ET-1 and AQP4,which would lead to reduction of blood-brain barrier permeability,alleviate the astrocyte cell edema induced by hypoxia and realize the prophylactic effect against hypoxic cerebral edema.This paper systematically studied and investigated the material basis and molecular mechanism of Tibetan Turnip about its "tonic and anti-hypoxia" beneficial effect and prophylactic effect against acute high altitude illnesses(hypoxic pulmonary/cerebral edema).It possessed important theoretical meanings and realistic values for Tibetan Turnip as functional food.