| Objective: To investigate the role of inducible co-stimulator(ICOS)in endothelial mesenchymal transition(End MT)and sclerosis in spontaneously hypertensive rats(SHR).Methods:1.There are 20 SHRs(observation group)and 20 Wistar-Kyoto rats(WKY,control group)of the same strain,all males,4 weeks old,were divided into 4 groups of 5 rats each using the random number table method.Changes in blood pressure and body weight were recorded from each group of rats using a non-invasive blood pressure monitor and a body weight monitor,and tissues associated with the rat mesentery were removed for experimental manipulation at 4 weeks,6 weeks(initial phase of hypertension),10 weeks(development of hypertension)and 30 weeks(target organ damage phase).The m RNA expression levels of ICOS,ICOSL,IL-21,TGF-β1,Collagen-I,Collagen-III,VE-cadherin and α-SMA were measured by RT-PCR in the mesenteric tissues of each group of rats.Protein distribution and expression of collagen-I,collagen-III,VE-cadherin,α-SMA and ICOS,ICOSL,IL-21 and TGF-β1 in mesenteric arterial vessels of rats in each group by immunohistochemical staining(IHC).The morphological structure and collagen fibrils in rat mesenteric arterial vessels were observed by HE and Masson’s staining.The expression levels of Collagen-I,Collagen-III,VE-cadherin,α-SMA and ICOS,ICOSL,IL-21 and TGF-β1in rat mesenteric lymphatic tissue were measured by Western blot.Changes in plasma levels of IL-21 and TGF-β1 in each group of rats were measured by enzyme-linked immunosorbent assay(ELISA).2.Based on method 1,10-week-old SHRs and WKY rats were selected for the study,with WKY rats as the control group(n=5)and SHRs divided into model group(SHR-A group,n=5)and ICOS signal blocking group(SHR-B group,n=5)according to the random number table method.The blocking group was injected once a week with100μL of ICOS blocking monoclonal antibody(25μg/100μL)by intraperitoneal injection,and the model group was injected once a week with 100μL of PBS solution(0.01mol/L)by intraperitoneal injection.The rats were injected continuously from 10 weeks of age for 20 weeks,and the changes in the corresponding indices in method 1were examined by RT-PCR,IHC,Western blot,ELISA,HE and Masson’s at 30 weeks in each group of rats.Result:1.Blood pressure values were significantly higher in SHRs compared with WKY rats from 6 weeks of age(P<0.05 or P<0.01)and body weight values were significantly lower from 20 weeks of age(P<0.05 or P<0.01).The m RNA expression levels of ICOS,ICOSL,IL-21,TGF-β1,Collagen-I,Collagen-III and α-SMA in the mesenteric tissues of SHRs from 6 weeks of age were significantly higher(P<0.05 or P<0.01)compared with those of WKY rats at the same time,the expression levels of VE-cadherin The expression levels of VE-cadherin decreased(P<0.05 or P<0.01).IHC staining showed that the expression of Collagen-I,Collagen-III and α-SMA gradually increased in the mesenteric micro-artery vasculature of SHRs,and their mean optical density values were significantly higher(P<0.05 or P<0.01)than those of WKY rats from 10 weeks of age.Similarly,30-week-old SHRs showed strong positive expression of collagen-I,collagen-III,α-SMA in the small mesenteric artery vessels and ICOS,ICOSL,IL-21,TGF-β1 in the lymphoid tissue.This corresponded to significantly higher mean optical density values than in WKY rats(P<0.01).The mean optical density values of VE-cadherin were lower than those of WKY rats from 10 weeks of age(P<0.01),and the mean optical density values of VE-cadherin in small arterial vessels were significantly lower than those of WKY rats at 30 weeks of age(P<0.01).ELISA showed that plasma levels of TGF-β1 and IL-21 were significantly higher in SHRs compared to WKY rats from 6 weeks of age(P<0.05 or P<0.01).HE and Masson’s staining showed a progressive thickening of the mesenteric artery wall in SHRs with the progression of hypertension and a deepening of arterial vascular fibrosis compared with WKY rats(P<0.05 or P<0.01).Western blot showed that the expression levels of TGF-β1 and IL-21 in mesenteric lymphatic tissues were significantly higher in SHRs compared with WKY rats from 6 weeks of age(P<0.05 or P<0.01),and the expression levels of Collagen-I,Collagen-III,α-SMA,ICOS and ICOSL in mesenteric arterioles and lymphatic tissues were significantly higher in SHRs compared to WKY rats from 10 weeks of age(P<0.05 or P<0.01).α-SMA,ICOS and ICOSL protein expression levels were also significantly higher than those of WKY rats(P<0.05 or P<0.01),whereas VE-cadherin protein expression levels were significantly lower than those of WKY rats from 10 weeks of age(P<0.01).Correlation analysis showed that the m RNA and protein expression levels of ICOS in mesenteric tissues of SHRs were significantly and positively correlated with the m RNA and protein expression levels of Collagen-I,Collagen-III,α-SMA and collagen volume ratio in mesenteric arterial vessels,while they were negatively correlated with the expression levels of VE-cadherin protein in blood vessels.2.After 20 weeks of ICOS signaling blockade,the blood pressure of rats in the SHR-B group was significantly lower(P<0.05)than that of rats in the SHR-A group but higher(P<0.05)than that of rats in the WKY group,while the body weight of rats in the SHR-B group had increased significantly(P<0.05)and was not significantly different from that of the WKY group.The results of RT-PCR,IHC staining and Western blot all showed that the m RNA and protein expression levels of Collagen-I,Collagen-III,α-SMA,IL-21 and TGF-β1 in mesenteric arterial vessels and mesenteric lymphatic tissues of rats in the SHR-B group were significantly lower(P<0.05)compared with those in the SHR-A group after 20 weeks of blockade intervention.The m RNA and protein expression levels of VE-cadherin were decreased(P<0.05)and upregulated(P<0.01)compared with those of rats in the SHR-A group,but some of the correlation indices were statistically different from those of rats in the WKY group.ELISA showed that the plasma levels of IL-21 and TGF-β1 were significantly lower in the SHR-B group compared with the SHR-A group(P<0.05),and not significantly different from the WKY group.After 20 weeks of blockade intervention,the wall thickness of mesenteric microartery and small artery vessels was moderate in the SHR-B group compared with the SHR-A group,and the abnormal proliferation of collagen fibers was significantly improved and the corresponding collagen volume ratio was significantly lower(P<0.05).Conclusion:1.The expression of ICOS is closely related to the occurrence and development of End MT and fibrosis processes in the mesenteric arteries of spontaneously hypertensive rats.2.Blocking the ICOS signal may potentially improve structural lesions in the mesenteric arterial vasculature and delay further worsening of hypertension by downregulating the expression of IL-21.Figure [32] Table [2] Reference [64]... |
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