Study On The Phenomenon Of Extrahepatic Pathological Angiogenesis In Multiple Organs Of HPS Rats And The Effect Of Adenosine A3 Receptor

Background and objectiveChronic liver disease（CLD）can lead to damage to multiple organs outside the liver（heart,lung,kidney,brain,etc）.More and more evidence that vasodilators and pro-angiogenic factors are significantly increased in chronic liver disease,resulting in abnormal angiogenesis in multiple organs.The pathological angiogenesis is closely related to the damage of organ function,suggesting that"extrahepatic pathological angiogenesis（EPA）"co-occurring in these organs may be one of the common pathological mechanisms of multiple organ damage in CLD.Further study on it is a new strategy for prevention and treatment of multiple organ function damage in patients with CLD.It has been reported in previous literature that pathological pulmonary angiogenesis is an important cause of hepatopulmonary syndrome（HPS）,which has become a new target for intervention research.Some foreign teams have reported that the common bile duct ligation（CBDL）rat model also has an EPA phenomenon in the intestine.In this study,we also accidentally found that extrahepatic angiogenesis may also exist in the cerebral cortex,kidney,and intestine,and may be related to the damage of extrahepatic organ function,which is worthy of further study.Therefore,the purpose of this study was to investigate the CBDL rat model:1.whether there is angiogenesis and functional damage in extrahepatic organs（such as lung,kidney,brain,intestine,etc.）;2.whether the vascular endothelial growth factor（VEGF）family mediates the EPA of CLD;3.whether A3 adenosine receptor（A3R）mediates the formation of EPA in the cerebral cortex.Materials and methodsIn this study,firstly,the rat model of CBDL was established,the microvascular density（MVD）was quantified by immunofluorescence staining,and the changes of MVD in different organs were compared.Then,the changes of VEGF family vascular factors in serum and organs were detected.The relationship between MVD and VEGF family vascular factors was analyzed.Finally,it is proved whether A3R mediates cerebral cortex angiogenesis in CBDL rats.The details are as follows:In experiment 1,the CBDL rat model was established.SD rats were divided into sham operation group（Sham group）and 5 weeks after CBDL operation group（CBDL 5w group）.After sampling,blood gas,serological,and urine indexes were detected.the morphological manifestations of various organs（liver,lung,brain,spleen,intestine,kidney）were observed,and the degree of organ damage was scored.In experiment 2,the angiogenesis of each organ of CBDL rats was evaluated by immunofluorescence staining,and the content of the VEGF family（VEGF,VEGFR1,VEGFR2,s Flt-1,and PLGF）in serum and organs were measured by ELISA kit.In experiment 3,the localization and relative expression of A3R in the brain of CBDL rats were detected by immunofluorescence staining,and the changes of vascular endothelial proliferation and migration-related signal pathway proteins（A3R,VEGF,FAK,and Src）were detected by q PCR and Western blot.Evans blue（EB）dye was used to quantitatively detect changes in the permeability of the blood-brain barrier（BBB）in rats.The water maze test was used to test the changes of rat behavior.Results1.Jaundice and ascites appeared in CBDL rats after 5 weeks.The levels of Alanine aminotransferase（ALT）,Aspartate aminotransferase（AST）,Total bilirubin（TB）,and Alkaline phosphatase（ALP）in the CBDL 5w group were higher than those in the Sham group（P<0.001）,indicating that liver function was significantly impaired.In the CBDL 5w group,bile duct dilatation,cholestasis,uneven surface of the liver texture,and small nodules were observed,and obvious liver fibrosis was also observed by Sirius red staining,indicating the success of the liver cirrhosis model.HPS is a complication of liver cirrhosis,which is mainly characterized by oxygenation disturbance.In the CBDL 5w group,P_aO₂ decreased（P<0.001）and P_（A-a）O₂increased（P<0.001）,and the CBDL 5w group had spot-like changes in the lungs,some necrotic lesions,hematoxylin-eosin staining showed severe alveolar disorder,obvious inflammatory cell infiltration,and pulmonary microvascular expansion.It shows that the typical changes of hepatopulmonary syndrome have been successfully reproduced.The above indicated that the CBDL rat model was established successfully.2.In addition to the liver and lung injuries,hematoxylin-eosin staining showed that compared with the Sham group,the CBDL 5w group rats 1)neuronal injury and coagulative necrosis in brain tissue,and the number of normal neurons were reduced（P<0.001）;2)The arrangement of the villi in the intestine was disorderly,from tall columnar to short and wide,and the number of dilated and congested red blood cells in the villi increased（P<0.001）;3)The renal tubules were dilated,and the renal index increased（P<0.05）.Detection of renal function indicators found that except for the 24h urine volume was not statistically significant,others were statistically significant.Among them,sodium ion concentration,creatinine,and blood urea nitrogen changed in the same way in serum and urine samples.Compared with the Sham group,the sodium ion concentration in the CBDL 5w group decreased,while the creatinine and blood urea nitrogen increased（P<0.01）.The 24h protein in the CBDL 5w group also increased（P<0.01）.Cystatin C（Cys-c）,which indicated renal function damage,was also increased in the CBDL 5w group（P<0.01）.It is suggested that extrahepatic multiple organs damage in CBDL rats.3.In the CBDL 5w group,the angiogenesis in the mesenteric window was disordered,the tip became thinner,and the number increased（P<0.001）.Immunofluorescence staining of vascular marker（CD31）showed that compared with the Sham group,the CD31 staining of the liver,lung,cerebral cortex,intestine,and kidney were significantly increased in the CBDL5w group except for the spleen.Moreover,the MVD of each organ was also increased（P<0.001）.The changes of VEGF family factors（VEGF,VEGFR1,VEGFR2,s Flt-1）in serum and various organs were inconsistent,which may be related to the content of MVD in various organs.It is suggested that extrahepatic pathological angiogenesis in CBDL rats is directly related to the changes of the VEGF family in serum in vivo.4.Immunofluorescence staining showed that a large number of A3R gathered around the cortical vessels.Compared with the Sham group,the m RNA expressions of A3R,VEGF,FAK,and Src in the CBDL 5w group were up-regulated（P<0.01）,and the protein expressions of A3R,VEGF,p-FAK,and p-Src were increased（P<0.01）,indicating that A3R promotes vascular endothelial migration and proliferation through the canonical VEGF/FAK/Src pathway,resulting in angiogenesis.The permeability test also found that the content of EB in the cerebral cortex of CBDL rats was increased（P<0.01）,indicating that the permeability of the brain was significantly changed.The water maze test found that the learning and memory abilities of CBDL rats were decreased（P<0.05）.Conclusions1.The experimental HPS rats model was successfully established.2.The pathological changes of extrahepatic multiple organs in HPS rats suggest organ damage.At the same time,there is extrahepatic pathological angiogenesis,which is directly related to the VEGF family.3.A3R promotes cerebral angiogenesis in HPS rats by activating VEGF/FAK/Src pathway and is associated with impairment of BBB permeability and cognitive dysfunction.