A Study On Characteristics And Formation Mechanism Of Sublingual Venae In Different Portal Hypertension

BackgroundSublingual venae diagnosis is an important component of traditional tongue diagnosis. Because it is easy to check and reflects fast, it is also an important indications of human qi-blood, fluid-humor and static blood in clinical diagnosis. Recently, sublingual venae diagnosis became an important method to diagnose the cardiovascular disease, the digestive system disease and diabetes and other diseases. With long time clinical observation of patients with primary liver cancer and cirrhosis, it is found that a large percent of those patients are suffering sublingual venae expansion, morphology circle change and the color change to blue, purple and crimson. These changes are sensitive and significantly related to the progress of treatment and the prognosis of the patients. In Traditional Chinese Medicine, liver and tongue are deep related. Abnormalities of liver may reflect on the tongues with corresponding changes. Many researches suggest that abnormal formation of sublingual collateral vessels of these patients may be related to the increased stress of portal vein. However, the mechanism of abnormal sublingual venae was rarely reported. Previous researches reported that the increasing pressure of sublingual venae resulted in the abnormal sublingual venae. However, we found that primary hepatic carcinoma patients with portal vein pressure are more likely to have the sublingual collaterals abnormalities and the width of Sublingual collaterals vessels was related to portal vascular and certain local hemodynamic indicators directly. Meanwhile, the result that the pathological changes of sublingual collateral vessels and determined the stress of portal vein of 118 portal hypertension patients at surgery also confirm that sublingual collateral vessels varicose rate is 100% in the portal hypertension patients. Based on this, we put forward one hypothesis that sublingual collateral is a component of human vein system as well as portal vein, and there must be some relationship in pathology between them. However, the formation of portal hypertension has three types, and there are extrahepatic obstruction, intrahepatic blockages and extrahepatic intrahepatic compound jam. Different formation mechanism of portal hypertension would lead to the different change of sublingual collaterals. The following days, we discovered that there were different changes between patients with cirrhosis and patients with portal hypertension caused by tumor obstruction. Therefore, it is difficult to explain by passive expansion of sublingual venae. This suggests us that abnormal sublingual collateral vessels which are caused by liver portal hypertension have deeper material basis.With the developing research of modern cancer and molecular biology, molecular mechanism of angiogenesis has been gradually revealed. For the adults, physiological and pathological mechanisms of angiogenesis are the main blood vessel formation. Lots of elements are playing positive regulatory roles in angiogenesis. In factors that known in angiogenesis, the vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF) are the most important. Hypoxic response, mechanical stress and a variety of mechanisms are involved in the VEGF gene induction. VEGF adjust angiogenesis through VEGF/VEGFR signal passage. Recent studies found that fat inflammation factors and its receptor, Apelin/APJ path, also have close relations with the angiogenesis, and are looked as new targets of treatment to the portal hypertension. In recent years research has been confirmed that the lungs and spleen also exists angiogenesis in the process of portal hypertension. Therefore, envisaging under the "angiogenesis" theory, pay the focus on the perspective of angiogenesis to explore the possible molecular mechanism of abnormal sublingual collateral vessels in different portal hypertension models.ObjectiveTo observe the changes and study the formation mechanism of sublingual collateral vessels in three different portal hypertension models. And from the Angle of angiogenesis, explore the relationship between VEGF/VEGFR2, Apelin/APJ signaling pathways and abnormal sublingual collateral vessels mainly in order to provide data support for revealing the molecular mechanism of abnormal sublingual collateral vessels. Methods(1) Methods of making different portal hypertension models with dog. Beagle dogs were randomly divided into five groups. There are normal group, sham group, prehepatic portal hypertension group, intrahepatic portal hypertension group, and compound portal hypertension group. There were 6 dogs in each group. The portal veins of dogs were ligatured in prehepatic portal hypertension group. Canine model of intrahepatic portal hypertension was established by injecting dimethylnitrosamine (DMN) into portal vein once a week which would last for 7 weeks. The above two methods were used to make compound portal hypertension model together. During the process of each model production, portal pressure of dogs was determined with the bioelectricity recording system. After the dogs were sacrificed, The serum liver function was detected with reagent kits, and the liver pathological changes were observed with HE coloration.(2) Methods of observing the features of tongue collaterals in different portal hypertension models. The changes of sublingual collaterals form and color were detected by macroscopic observation. The width of tongue root collaterals was measured by vernier caliper. CD31 protein expression of sublingual collateral vessels was detected by immunohistochemical coloration. The morphological changes the endothelial cells of sublingual collaterals were observed by transmission electron microscope.(3) Methods of studying the formation mechanism of sublingual collateral vessels in different groups. Nitric oxide in serum was detected by nitrate reductase method. The vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α(HIF-1α) expression of serum was detected by ELISA. The proteins expression of VEGF, HIF-1α, VEGFR2, Apelin and APJ in sublingual tissues was detected by immunohistochemical coloration and western blotting. The mRNA expression level of VEGF, VEGFR2 and HIF-1αin sublingual tissues was detected by Real-Time PCR.Results(1) The portal vein pressure of dogs was beginning to rise after DMN induction 2 weeks in intrahepatic and compound groups compared with this in sham group(P<0.05). The portal vein pressures of dogs in model groups were a ladder shaped up in 4 weeks, six weeks, eight weeks with experimental progress(P<0.01),and were stable after 8 weeks. The pressure of dogs in compound group further increased after operation of portal vein ligation(P<0.01). After portal vein ligation, the pressure of dogs in prehapatic model group lifted to peal quickly, and gradually declined to a high level when the dogs were sacrificed(P<0.01).What's more, the dog anomalous changes of the liver function and liver pathology were similar with human.(2) The morphous and color of sublingual collateral vessels of the beagle dogs in intrahepatic and compound model groups changed obviously compared with those in normal control group and sham group. Sublingual collateral vessels appeared enlarged tortuous and increased diameters, the increased number of small collateral vessels and even bluish purple. While in prehepatic model group, sublingual collateral vessels appeared color changed only. Immunohistochemical coloration results showed that the expression of CD31 protein increased compared with this in normal and sham group (P<0.01), and the most obvious increase was in compound model group. Transmission electron microscopy showed that sublingual hemal walls of dogs in three model groups were thicken totally or partly, vascular cavity becomes small, new capillaries appeared, and membrane boundary of endothelial cell is not clear, cell nuclei lost or disappeared. For changes degree, it was heaviest in compound model group, second in intrahepatic model group, and lightest in prehepatic model group.(3) NO in serum of the beagle dogs was significantly increased in model groups compared with normal and sham groups (P<0.01). VEGF, HIF-1αprotein expression in serum was significantly higher in two model groups (P<0.01), while no significant changes in prehehatic model group. Immunohistochemical coloration results showed that there is almost no expression of VEGF, VEGFR2 and HIF-1αof sublingual collateral vessels in the normal control group. But the expression of three model groups increased. The mRNA of VEGF, VEGFR2 and HIF-1αalso significantly increased. As for Apelin and APJ, their protein expression was same as others. ConclusionThe dog sublingual collaterals vessels appeared macroscopic and microcosmic changes in three kinds of portal hypertension models. Portal hypertension caused by intrahepatic obstruction may be contributing to the formation of abnormal sublingual collaterals vessels. Extrahepatic jam often becomes the aggravation of factors inducing collaterals changes. Angiogenesis induced by VEGF and Apelin participated in the formation of abnormal sublingual collaterals. They reacted by HIF-1α/ VEGF/ VEGFR2/ NO and Apelin/APJ pathways respectively. It may be one of the important mechanisms of vascular remodeling in abnormal sublingual collaterals vessels.