| Objective:The blood stasis model rats were treated with Xuefu Zhuyu capsule,to analyze the effect of Xuefu Zhuyu capsule on blood flow and tissue angiogenesis.The blood flow rate response genes KLF2 and KLF4 and downstream key angiogenesis factors(VEGFA/VEGFR2 and e NOS)were detected.The effect of "promoting blood circulation and angiogenesis" of Xuefu Zhuyu capsule was comprehensively analyzed,in order to understand the role of KLF2/4 signal in this process.Methods:Forty SD rats were randomly divided into blank group,model group,conventional dose group and high dose group,with 10 rats in each group.The dose of Xuefu Zhuyu capsule was 0.48 g/(kg·d)in the conventional dose group,and 0.96g/(kg·d)in the high dose group,both of which were 20 m L/(kg·d).The blank group was given normal saline at 20 m L/(kg·d),twice a day.(1)The contents of Xuefu Zhuyu capsule were dissolved in normal saline,the drug group was preventively administrated with Xuefu Zhuyu capsule for 11 days,blank group and model group were intragastric with equal amount of normal saline.(2)After the drug group received prophylactic administration for 11 days,except blank group,rats were subcutaneously injected with 0.1% epinephrine hydrochloride injection combined with ice water swimming for modeling.Continuous processing for 3 days to establish blood stasis model of rats.(3)One day before moulding and three days after moulding,automatic hemorheometer was used to detect hemorheological indexes of blood,and color Doppler ultrasound was used to detect blood flow rate of femoral artery of lower limb to evaluate whether the rats were modeled.(4)Bilateral hind limb amputation was performed on the first day after modeling,and granulation tissue and wound tissue were collected 48 hours after surgery.(5)HE staining and immunohistochemistry were used to detect the neovascularization of granulation tissue in each group.(6)The expressions of KLF2,KLF4 and VEGFA,VEGFR2 and e NOS in granulation tissues of rats in each group were detected by immunohistochemistry.Western Blot and Q-PCR were used to detect protein and m RNA expressions of VEGFA,VEGFR2,e NOS and KLF2 and KLF4 in wound tissues of rats in each group.Results:1.Observation results of blood stasis characterization in rats:Before and during prophylactic administration,rats in each group ate normally,their body weight increased at a uniform rate,and their paws and tails were light red.After modeling,the rats in the model group ate less than those in the blank group,the body weight ratio decreased significantly before and after modeling(P<0.01),paw and tail color was lighter and more purple.Compared with the model group,the body weight ratio of the drug group increased significantly before and after modeling(P<0.01),and claw,tail color was more red.2.Results of hemorheology and blood flow rate in rats:Compared with blank group,the preparation of blood stasis model made the indexes of hemorheology increase significantly in model group(P<0.01),and the blood flow rate of femoral artery in lower limb slowed down(P<0.01);Compared with the model group,the intervention of Xuefu Zhuyu capsule significantly improved the indexes of hemorheology(P<0.01),and increased the blood flow rate of femoral artery of lower extremities(P<0.01).3.Results of the angiogenesis test:(1)The HE staining results of granulation tissue showed that the number of blood vessels in granulation tissue of model group was significantly reduced compared with blank group(P<0.01);Compared with model group,the number of blood vessels in granulation tissue in both drug groups was significantly increased(P<0.01).(2)Immunohistochemical analysis of granulation tissue endothelial cell marker CD34 showed that the expression of CD34 in model group was lower than that in blank group(P<0.05).Compared with model group,the expression of CD34 in routine dose group and high dose group was significantly increased(P<0.05).4.Detection results of flow rate response transcription factor KLF2/4 expression:Immunohistochemistry of granulation tissue,q-PCR and Western Blot results of wound tissue showed that compared with blank group,kl F2/4 expression was significantly up-regulated in model group(P<0.05),and significantly down-regulated in both drug groups compared with model group(P<0.05).5.Expression of kl F2/4-regulated target genes VEGFA,e NOS and VEGFR2:Immunohistochemistry of granulation tissue,q-PCR and Western Blot results of wound tissue showed that VEGFA,VEGFR2 and e NOS were down-regulated in model group compared with blank group(P<0.05).The expressions of VEGFA,VEGFR2 and e NOS were up-regulated in both drug groups compared with model group(P<0.05).Conclusion:1.For blood stasis model rats,Xuefu Zhuyu capsule has the effect of "promoting blood circulation and angiogenesis" by improving hemorheological indicators,increasing blood flow rate and promoting angiogenesis.2.The expression of KLF2/4 was inhibited in the process of "promoting blood circulation and angiogenesis" in xuefu Zhuyu capsule promoting blood stasis model rats,and the two were correlated.3.Klf2/4 may be involved in xuefu Zhuyu Capsule to “promote blood circulation and angiogenesis” in blood stasis rats by regulating VEGFA/VEGFR2,e NOS and other key signal molecules to promote angiogenesis. |
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