Study On The Protective Effects And Mechanisms Of Three Types Of Natural Products And Their Derivatives On Vascular And Tracheal Injur

Objective:Diabetes vascular complications(DMVC)is one of the most common and serious complications of diabetes.Ginsenoside comes from Chinese traditional medicine ginseng,while luteolin(LTD)is a flavonoid compound from many medicinal plants.According to the previous research and literature reports of the research group,ginsenoside,LTD and its derivative LTD2 have the function of protecting blood vessels,but their pharmacological effects are still unclear.Therefore,in this study,we used the model of high glucose(HG)-induced vascular injury in vitro,and the model of DM mice in vitro,and used the Wire Myograph microvascular tension measurement technology to study the protective effect and mechanism of ginsenoside,LTD and its derivatives on HG and DM-induced vascular endothelial injury.Methods:1.HG-induced vascular injury model experiment in vitro:isolate and prepare the isolated vascular rings of the basilar artery(BA)of SD rats,and screen the relaxation activity of ginsenoside,LTD and its derivatives on the BA vessels of SD rats using the Wire Myograph vascular tension measurement system,and then use 70mM HG+1 μM hydrogen peroxide(H2O2)combined with simulated pathological injury of DM(HG was incubated with H2O2 for 20 min at the beginning of incubation,and then replaced with HG at the end of incubation.The total incubation time was 4.5 h,and the temperature was 37℃).The vascular endothelial injury model of DMVC was induced in vitro.After the vessels were incubated with ginsenoside,LTD and its derivatives,the vasoconstrictor thromboxane receptor agonist(U46619)was used to constrict the vessels,Acetylcholine(Ach)-induced vascular endothelium-dependent relaxation dose-effect curve(Ach-dose effect curve)was measured by cumulative concentration administration to evaluate the vascular endothelium-dependent relaxation effect(vascular endothelial relaxation function).Then,the blood vessels were incubated with nitric oxide synthase(NOS)inhibitor(LNAME:non-selective inhibition of NOS)and guanylyl cyclase(sGC)inhibitor(ODQ,selective inhibition of sGC)to explore the relationship between the pharmacological mechanism of ginsenoside,LTD and their derivatives on vasodilation and eNOS and sGC.2.In vitro vascular experiment of DM mice:type 1 diabetes(T1DM)mouse model was established by intraperitoneal injection of STZ(50mg/kg)for 5 days,and type 2 diabetes(T2DM)mouse model was established by intraperitoneal injection of STZ(50mg/kg)for 5 days after feeding with high-fat diet for 8 weeks.T1DM mice were fed for 10w with LTD and LTD2(45 and 90mg/kg)for 8w,and T2DM mice were fed for 6w with LTD and LTD2(45 and 90mg/kg)for 13w,respectively.After the animals were killed,the thoracic aorta(TA)vessels were isolated.Wire Myograph vascular tension measurement system was used,and phenylephrine(Phe),a vasoconstrictor,was used to constrict the blood vessels.Ach-induced endothelium-dependent vasodilation dose-response curve(Ach-dose-response curve)was measured by cumulative concentration administration,and the effects of LTD and LTD2 on TA endothelium-dependent vasodilation in T1DM and T2DM mice were observed.In addition,the levels of TA vasoactive oxygen species(ROS)in T1DM and T2DM mice were measured by freezing section and dihydroethidium(DHE)staining to study the effects of LTD and LTD2 on vascular oxidative stress.Results:一.Experimental results of HG-induced vascular injury model in vitro1.The effect of ginsenoside on vasoconstriction and vasodilation in vitro and HG-induced vascular injury in vitro1.1 Relaxation effect on U46619 induced vasoconstrictionOn the isolated blood vessels of normal BA,compounds 20R-Protopanaxatriol and R-Rg3 had a relaxing effect on the vasoconstriction induced by U46619,Emax was 47.29±2.76%and 65.94±4.36%,respectively,EC50 was 3.07 ± 1.43μM and 3.44 ±1.09μM.1.2 Effect on the dose-response curve of U46619 induced vasoconstriction20R-Protopanaxatriol and R-Rg3 have a relaxing effect on the isolated BA vasoconstriction induced by U46619.With the increase of the concentration of 20R-Protopanaxatriol and R-Rg3,the U46619-dose-effect curve gradually moves downward,of which 100μM 20R-Protopanaxatriol and 60μM R-Rg3 has the strongest effect on vasoconstriction.1.3 Effect on HG-induced vascular injury in vitroCompared with the Normal group,the Ach-dose-effect curve of BA vessels in the Model group moved up significantly,Emax decreased significantly,and EC50 increased slightly.Compared with the model group,the Ach-dose-response curve of the 20R-Protopanaxatriol and R-Rg3 groups significantly moved down,Emax significantly increased,and EC50 slightly decreased,suggesting that ginsenoside has a certain protective effect on HG-induced vascular injury in vitro.1.4 Relationship between vasodilation and endothelium of tested compounds20R-Protopanaxatriol has a relaxing effect on isolated vessels with intact endothelium,but has no relaxing effect on isolated vessels without endothelium;R-Rg3 has a relaxing effect on both intact and de-endothelized vessels,suggesting that 20R-Protopanaxatriol vasodilation depends on endothelium,while R-Rg3 does not depend on endothelium.1.5 The relationship between 20R-Protopanaxatriol antagonizing HG-induced vascular injury in vitro and NOS,sGCCompared with the Normal group,the Ach-dose-effect curve of blood vessels in the Model group was significantly higher,Emax was significantly lower,and 20R-Protopanaxatriol significantly reversed the Ach-dose-effect curve of blood vessels in the Model group.After LNAME and ODQ treatment,the Ach-dose-effect curve of blood vessels in Normal and Model groups significantly moved up,Emax significantly decreased,and EC50 also increased.The results showed that LNAME and ODQ weakened the vasodilation effect of 20R-Protopanaxatriol,which was related to NOS and sGC.2.The effect of LTD and LTD2 on the vasomotor response and HG-induced vascular injury in vitro2.1 Relaxation effect on U46619 induced vasoconstrictionOn normal BA isolated blood vessels,LTD and LTD2 had a relaxing effect on the vasoconstriction induced by U46619,Emax was 52.28±3.90%and 49.74±4.67%respectively,EC50 was 25.85±5.90μM and 21.43±4.76μM.2.2 Effect on the dose-response curve of U46619 induced vasoconstrictionLTD and LTD2 have a relaxing effect on the vasoconstriction effect induced by U46619.With the increase of the concentration of LTD and LTD2,the dose response curve of U46619 gradually moves down and presents a concentration dependence,of which 30μM LTD and 100μM LTD2 significantly antagonized vasoconstriction and Emax significantly increased.2.3 Effect on HG-induced vascular injury in vitroCompared with the Normal group,the Ach-dose-effect curve of BA vessels in the Model group moved up significantly,Emax decreased significantly,and EC50 increased slightly.Compared with the model group,the Ach-dose-response curve of the LTD and LTD2 groups was significantly lower,Emax was significantly increased,and EC50 was slightly decreased,suggesting that the LTD and LTD2 had a certain protective effect on HG-induced vascular injury in vitro.2.4 Relationship between vasodilation and endothelium of tested compoundsLTD and LTD2 have a relaxation effect on the isolated vessels with intact endothelium,but no relaxation effect on the isolated vessels without endothelium.The Ach-dose-effect curve of the vascular ring with endothelium removed moved up significantly,while Emax decreased significantly.The results suggest that the vasodilation effect of LTD and LTD2 depends on vascular endothelium.2.5 Correlation between the antagonism of LTD and LTD2 to HG-induced vascular injury in vitro and NOS and sGCCompared with Normal group,the vascular Ach-dose-response curve of Model group moved up,Emax decreased significantly,and the vascular Ach-dose-response curve of Model group was significantly reversed by LTD and LTD2.After LNAME and ODQ treatment,the vascular Ach-dose-response curve of model group significantly moved up,Emax significantly decreased,and EC50 also increased.The results showed that LNAME and ODQ weakened the vasodilation of LTD and LTD2,and the vasodilation of LTD and LTD2 was related to NOS and sGC.二.Results of vascular experiment in vitro in DM mice1.Effect of LTD and LTD2 on TA vascular endothelium-dependent relaxation function in T1DM and T2DM mice.Compared with Normal group,the Ach-dose-response curve of TA vessels in T1DM and T2DM mice significantly moved up,Emax decreased and EC50 increased.After intragastric administration of LTD and LTD2,the Ach dose response curve of TA blood vessels significantly moved downward,Emax increased,and EC50 decreased,suggesting that LTD and LTD2 can improve TA vascular diastolic function in T1DM and T2DM diabetes mice.2.Effect of LTD and LTD2 on TA vascular oxidative stress in T1DM and T2DM miceCompared with Normal group,the fluorescence intensity and expression of DHE staining in TA vessels of T1DM and T2DM mice were significantly increased.Compared with the model group,the fluorescence brightness and fluorescence intensity of DHE staining in TA blood vessels of mice in LTD and LTD2 groups were significantly decreased.Conclusion:1.In the experiment of HG-induced vascular injury in vitro,HG significantly reduced the endothelium-dependent relaxation function of the isolated vessels.20R-Protopanaxatriol,R-Rg3,LTD,LTD2 antagonize HG-induced vascular injury in vitro.In addition,the effect of these compounds on improving HG-induced vascular injury in vitro was antagonized by LNAME and ODQ,suggesting that their mechanism of action was related to NOS/sGC.2.In the DM-induced DMVC experiment in mice,both T1DM and T2DM significantly reduced the endothelium-dependent relaxation function of blood vessels,increased the ROS level of blood vessels,and induced vascular endothelial and oxidative stress damage.LTD and LTD2 can improve vascular endothelial relaxation function,antagonize vascular endothelial injury induced by DM,significantly reduce vascular ROS level,and inhibit vascular oxidative stress injury induced by DM.Objective:The contraction and relaxation of tracheal smooth muscle play a key role in the pathogenesis of many respiratory diseases.The discovery of compounds that can regulate the contraction and relaxation of tracheal smooth muscle has important significance and value in the treatment and prevention of respiratory diseases.Ginsenoside comes from the traditional Chinese medicine ginseng.Forskolin(FSK)and Isoforskolin(IS OF)are diterpenoid compounds extracted from the plant of Coleus forskolin.The existing literature and the previous research of the research group found that FSK and IS OF can improve the lung function of rats/mice with chronic obstructive pulmonary disease(COPD),ISOF has both anti-inflammatory(including anti-acute and chronic inflammation)and tracheal dilation pharmacological effects,and ginsenoside also has anti-inflammatory pharmacological effects,but its regulation and mechanism of action on tracheal smooth muscle are not clear,so this study is on guinea pig isolated trachea,The antagonistic effect of ginsenoside,ISOF and FSK on the contraction of isolated guinea pig tracheal rings induced by histamine(His)and acetylcholine(Ach)was studied by using the wire myograph microvascular tension measurement technology,and the effect and mechanism of ginsenoside,ISOF and FSK on the relaxation of isolated guinea pig tracheal rings were clarified.Methods:Separate and prepare the isolated air tube ring of guinea pigs,give 15mN pretension and balance for 1h at 37℃,during which time,continuously pass compressed air,and change the working fluid every 20min.Then use 60mM of MOPS-K+PSS to stimulate the trachea for 5-10 minutes,wash the trachea with MOPS-PSS for 3 times,and repeat the operation with MOPS-K+PSS for a total of 2 times.When the difference between the two trachea contractions is less than 10%,the trachea activity is qualified.30μM His or 100μM Ach induced tracheal contraction.After reaching the maximum contraction,the cumulative concentrations of ISOF,FSK,ginsenoside and their derivatives were given,and the dose-response curves were observed and Emax and EC50 were calculated.Results:1.Effect of ginsenoside and its derivatives on the contraction of guinea pig trachea induced by AchGinsenoside has a relaxing effect on the contraction of isolated guinea pig tracheal smooth muscle induced by Ach.Among them,20R-Protopanaxatriol,20S-Protopanaxatriol and 20R-Panaxatriol have stronger relaxation effect on the contraction effect of isolated guinea pig tracheal smooth muscle induced by Ach than Rbl,R-Rg3,R1 and Rg1.20R-Protopanaxatriol has the strongest relaxation effect on the contraction effect of isolated guinea pig tracheal smooth muscle induced by Ach,with its Emax of 51.60±6.59%and EC50 of 3.07 ± 1.43μM.2.Effect of ginsenoside and its derivatives on His-induced tracheal contraction in guinea pigsGinsenoside has a relaxing effect on the contraction of isolated guinea pig tracheal smooth muscle induced by His.Among them,20R-Protopanaxatriol,20S-Protopanaxatriol and 20R-Panaxatriol have stronger relaxation effect on the contraction effect of isolated guinea pig tracheal smooth muscle induced by His than Rbl,R-Rg3,R1 and Rg1,and 20R-Panaxatriol has the strongest relaxation effect on the contraction effect of isolated guinea pig tracheal smooth muscle induced by His,with Emax of 76.40±5.65%and EC50 of 8.43 ± 2.20μM。3.Effect of FSK and ISOF on the contraction of guinea pig tracheal smooth muscle induced by AchOn isolated guinea pig tracheal smooth muscle,FSK and ISOF had a relaxant effect on Ach induced contraction of isolated guinea pig tracheal smooth muscle,with Emax of 53.60±3.89%,37.18±2.80%,and EC50 of 11.29±3.05 μM、20.80±6.03 μM。4.Effect of FSK and ISOF on His-induced contraction of guinea pig tracheal smooth muscleOn the isolated tracheal smooth muscle of guinea pigs,FSK and ISOF had a relaxing effect on the contraction of isolated tracheal smooth muscle of guinea pigs induced by His,with Emax of 145.60±5.97%and 139.07±7.08%,respectively.EC50 of 1.24 ±0.41μM and 2.78±0.65μM.Conclusion:1.Ginsenoside has a relaxing effect on the contraction of isolated guinea pig tracheal smooth muscle induced by Ach or His,and 20R-Protopanaxatriol,20S-Protopanaxatriol,and 20R-Panaxatriol have a more significant antagonistic effect on His induced contraction of isolated guinea pig tracheal rings than Rbl,R-Rg3,R1,and Rg1.FSK and IS OF have a relaxing effect on the contraction of isolated guinea pig tracheal smooth muscle induced by Ach or His,while FSK and IS OF have a stronger inhibitory effect on the contraction of isolated guinea pig tracheal smooth muscle induced by His.