Effects And Molecular Mechanisms Of Taoren On Blood Circulation Disorder Induced By Different Factors In Rats

The blood circulation disorder (BCD) is a pathophysiological state induced by local or systemic circulatory inadequacy. It can also result in tissue ischemia hypoxia injury, metabolic disorder, organ failure, even life-threatening. Microcirculation disturbance (MCD), which occurs in the local micro-vascular blood circulation, is an important component of BCD. BCD-related disease has been the thorny problem of therapy.Cold stagnation and blood stasis syndrome and heat stagnation and blood stasis syndrome (HS and RS), has been found with characteristics of BCD. Traditional Chinese medicine has achieved ideal effects on these two syndromes. So looking for a safe and effective traditional Chinese medicine to prevent BCD-related disease has great significance, as well as studying on its efficacy characteristics and mechanisms.Taoren, one kind of traditional Chinese medicine, is the seed of heart. It has been found with effects of blood vessel dilation, increasing organ blood flow, inhibiting platelet aggregation, anticoagulation, anti-thrombosis, and so on. Our previous studies found that Taoren act effectively on improving the two syndromes. Therefore, these study committed to find the effects, mechanisms and the differences of Taoren on BCD induced by the two syndromes in rats. The study was divided into three parts:Part I:Study on the Characteristics of Blood Circulation Disorder in HS and RS RatsObjective:To study the different characteristics of BCD in HS and RS rats.Methods:The rats were randomly divided into 4 groups,10 rats in each group:normal control group of HS (HN), model group of HS (HM), normal control group of RS (RN), model group of RS (RM). HM group rats were put into the -18±2℃freezer for 2 hours, two times a day for seven consecutive days. To RM rats, Carrageenan solution was given intraperitoneally for six consecutive days, after that dry yeast solution was given to them subcutaneous ly on the seventh day. On the eighth day, all of the rats were detected the following indexes:(1) the Chinese medicine symptoms, (2) blood flow velocity (Fve) and blood flow score (Fsc), (3) blood viscosity (Vis) and blood fibrinogen (Fib), (4) arteriole and small artery diameter (Adia, s-Adia), venular and small vein diameter (Vdia, s-Vdia), glomerulus area (Gare), thrombosis rate (Trat), organ injury severity score (Isco) (including heart, lung, liver, kidney, spleen) by histopathology analysis (HE, PASM and PTAH staining).Results:(1) The corresponding Chinese medicine symptoms appeared respectively in both HM and RM rats. (2) The decreased blood flow velocity appeared in both HM and RM rats, while the decreased blood flow score appeared just in RM rats. (3) The increased blood viscosity appeared in both HM and RM rats, while the increased blood fibrinogen appeared just in RM rats. (4) The increased small artery diameter appeared just in HM rats. The increased glomerulus area appeared in both HM and RM rats. The arteriole, venular and small vein diameter did not change significantly in both HM and RM rats. The increased thrombosis rate and increased organ injury severity score appeared respectively in both HM and RM rats, in addition to the lung injury severity score in RM rats.Conclusion:There was BCD in rats with cold stagnation and blood stasis syndrome and heat stagnation and blood stasis syndrome, induced by frozen method and Carrageenan respectively. Its systemic manifestations included decreased blood flow velocity and increased blood viscosity. Its local manifestations included changes of vascular diameter, thrombosis, organ injury. The differences between HS and RS included blood flow score, blood fibrinogen, small artery diameter and the range of organ injury.Part II:Effects of Taoren on Blood Circulation Disorder in HS and RS RatsObjective:To study the different effects of Taoren on BCD in HS and RS rats.Methods:The rats were randomly divided into 2 part,4 groups in one part, 10 rats in each group, the 1st 4 groups:normal control group of HS (HN), model group of HS (HM), Taoren treatment group of HS(HT), Chuanxiong control group of HS(HC), the 2nd 4 groups:normal control group of RS(RN), model group of RS (RM), Taoren treatment group of RS(RT), Danshen control group of RS(RD). HM, HT and HC rats were put into the -18±2℃freezer as Part I. The carrageenan and dry yeast solution were given to RM, RT and RD rats as PartⅠ. At the same time, the corresponding traditional Chinese medicines were given to HT, HC, RT and RD rats for sever consecutive days. On the eighth day, all of the rats were detected the following indexes:(1) blood flow velocity (Fve) and blood flow score (Fsc), (2) blood viscosity (Vis) and blood fibrinogen (Fib), (3) small artery diameter (s-Adia), glomerulus area (Gare), thrombosis rate (Trat), organ injury severity score (Isco) (including heart, lung, liver, kidney, spleen) by histopathology analysis (HE, PASM and PTAH staining).Results:The effects of Taoren on BCD included:(1) The increased blood flow velocity appeared in both HT and RT rats, while the blood flow score did not change in both HT and RT rats. (2) The decreased blood viscosity appeared in both HT and RT rats, while the blood fibrinogen did not change in both HT and RT rats. (3) The decreased small artery diameter appeared just in HT rats, while the increased small artery diameter appeared in RT rats. The decreased glomerulus area appeared in RT rats, while the glomerulus area did not change in HT rats. The thrombosis rate did not change in both HT and RT rats. The decreased kidney injury severity score appeared in both HT and RT rats, while the decreased injury severity score of heart, lung and liver appeared just in HT rats. The spleen injury severity score did not change in both HT and RT rats.Conclusion:Taoren improved BCD in rats with HS and RS. The improvements lied in increasing blood flow velocity, decreasing blood viscosity, regulating the local vascular diameter and protecting kidney from injury. The different effects of Taoren on the two syndromes included:decreasing small artery diameter and protecting more organs from injury in HS rats, but increasing small artery diameter and protecting only for kidney from injury in RS rats.PartⅢ:Molecular Mechanisms of Taoren on Blood Circulation Disorder in HS and RS RatsObjective:To study the different molecular expression of HS and RS, the molecular mechanisms of Taoren on BCD in the two syndromes.Methods:The organ specimens were derived from PartⅡ. All of the specimens were detected the molecular expression by histopathology analysis (IHC, TUNEL, ISH). The indexes included:(1) adhesion molecule CD31 and vascular endothelial cell (VEC) growth factor VEGF, (2) VEC apoptosis, apoptotic inhibitor Bcl-2, apoptotic induced protein P53, (3) nucleus factor NFκB p65 and mRNA, inhibitor IκB-a and mRNA, (4) hepatic CD68+ macrophage and Caspase-1 P20 protein, (5) vascular pericyte (VPC) protein a-SM-actin, vascular smooth muscle cell (VSMC) receptor AT1 and ADRB2.Results:Molecular expression in HM and RM rats:(1) Increased CD31 expression appeared just in RM rats, while it did not change in HM rats. VEGF expression did not change in both HM and RM rats. Increased apoptosis appeared in both HM and RM rats. Decreased Bcl-2 expression appeared just in RM rats, while it did not change in HM rats. Decreased P53 expression appeared just in HM rats, while it did not change in RM rats. It was decreased NFκB protein, increased its mRNA and without its nucleus expression in HM rats. While in RM rats, it was decreased NFκB protein, increased its nucleus expression and without its mRNA change. It was increased IκB mRNA, without change on its protein and its nucleus expression in HM rats. While in RM rats, it was increased nucleus expression, without change on its protein and its mRNA. (2) Decreased hepatic CD68+ macrophage appeared just in HM rats, while it did not change in RM rats. Caspase-1 P20 protein did not change in both HM and RM rats. (3) Decreased ADRB2 expression appeared in both HM and RM rats, while expression of a-SM-actin and AT1 did not change in the two models.The changes of molecular expression in HT and RT:(1) Increased CD31 expression appeared just in HT rats, while it did not change in RT rats. Decreased VEGF expression appeared just in RT rats, while it did not change in HT rats. Apoptosis did not change in both HT and RT rats. Increased Bcl-2 expression appeared just in RT rats, while it did not change in HT rats. P53 expression did not change in both HT and RT rats. It was increased NFκB nucleus expression, without change on its protein and its mRNA in HT rats. While in RT rats, it was increased NFκB protein and its mRNA, without change on its nucleus expression. It was decreased IκB protein, without its nucleus expression and its mRNA change in HT rats. While in RT rats, it was decreased nucleus expression, without change on its protein and its mRNA.Conclusion:(1) Molecules closely related to HS included P53, NFκB, IκB and ADRB2. The promoting factors of HS might be the increased P53-induced apoptosis of endothelial cells and the decreased repair capacity of macrophages. The serious injury of endothelial cells and smooth muscle cells, caused by repeated freezing and rewarming, might be the reason of decreased activity of NFκB and ADRB2.(2) Molecules closely related to RS included CD31, Bcl-2, NFκB, IκB and ADRB2. The promoting factors of RS might be the increased CD31-induced adhesion and Bcl-2-related apoptosis of endothelial cells, besides of the increased inflammatory factor induced by activated NFκB.(3) Molecules affected by Taoren in HS included CD31, NFκB, IκB and AT1. The inhibited factors of circulation recovery might be increased CD31 and activated NFκB. The acceleration of circulation recovery might be the vascular contractile response induced by AT1 and the increased repair capacity of macrophages.(4) Molecules affected by Taoren in RS included VEGF, Bcl-2, NFκB and IκB. The persistent factors of apoptosis might be the decreased VEGF and increased Bcl-2 of endothelial cells. The persistent activity and increased transcription of NFκB might be closely related to the decreased activity of IκB. These might not be conductive to the recovery of BCD, and remained to be in-depth study.