The Molecular Mechanism Of Microvessel Proliferation And Basal Membrane Degradation In Rats Under Hypoxia

Plateau is a special natural environment,which is characterized by low pressure and low oxygen.A series of clinical syndroms that is characterized by excessive erythrocyte hyperplasia and hypoxemia,occur when people living in the plateau gradually lose their habit to the hypoxic environment of the plateau for a long time,which seriously affects the health of people in the plateau.Due to the excessive accumulation of red blood cells in the body at high altitude and low oxygen state for a long time,the blood viscosity increases and the blood flow slows down,and the body develops microvascular hyperplasia to improve blood circulation and compensate for oxygen supply,but the mechanism of which has not yet been clarified.IL-6 /JAK2/STAT3 pathway is involved in the regulation of angiogenesis to promote the metastasis and progression of tumor under local hypoxia condition in neoplastic diseases.under hypoxic condition,the secretion of IL-6 increased,which further regulated the expression of P-JAK2 and P-STAT3 which are the downstreams of JAK2/STAT3.In a series of ischemic hypoxic diseases,the high expression of MMP-9 promotes the regeneration of microvessels and basal membrane degradation.However,JAK2/STAT3 pathway and MMP-9 are very important factors in microvessel angiogenesis and basal membrane degradation,and which have been extensively studied in tumor angiogenesis.However,it is unclear how does the IL-6 /JAK2/STAT3/ MMP-9 pathway change in the bone marrow under long-term hypoxia and whether it is involved in microvascular proliferation and basal membrane degradation.Vascular basement membrane is a kind of complex dense mesh structure and its components include: layer adhesion proteins,IV type collagen protein and fiber connection,etc.MMP-9's main biological function is IV type collagen degradation.under hypoxic condition,the high expression of MMP-9 leads to the degradation of the basal membrane of blood vessels.COL4A1,as an important IV type collagen fiber,it is not clear that how does it lead to basement membrane degradation under MMP-9 regulation.Part I The research on microvessel changes in the bone marrow of rats are related to the IL-6/JAK2/STAT3/MMP-9 pathway under chronic hypoxia.Objective The microvascular is the important structural basis on the microenvironment in the bone marrow which is the main part of adult hematopoietic.The JAK/STAT3/MMP-9 are involved in the pathophysiological process of promoting microangiogenesis.However,the JAK2/STAT3's effect on regulating MMP-9 need to be further verified.At present,whether the IL-6 /JAK2/STAT3/MMP-9 pathway regulates hypoxic-induced microvascular proliferation and basal membrane degradation has not been studied.This research include the test of HB,HCT,RBC in peripheral blood,the level of IL-6 in serum and P-JAK2,P-STAT3 and m RNA and protein level of MMP-9,microvascular density and basement membrane degradation in the bone marrow comparing with the control group and with the application of STAT3 inhibitor,further verification of the above indicators to find the relationship between them.To investigate the role of IL-6 /JAK2/STAT3/MMP-9 pathway in bone marrow microvascular hyperplasia and basal membrane degradation.Methods The rats model were established by deviding 120 male SD rats into control group,chronic hypoxia group and chronic hypoxia+inhibitor(inhibitor SH-4-54 using lavage for 30 days,7.5 mg/kg/d)and chronic hypoxia+Solvent group(the final concentration of DMSO is 1.3%)randomly.The rats of hypoxia groups were prepared in low pressure oxygen cabin for medical research center,by simulating at an altitude of 5000 meters,24 h/day for 30 consecutive days.After 30 days,blood routine tests were performed.The serum IL-6 level of rats in each group was determined by Elisa.The research detect the level of MMP-9 m RNA by RT-PCR and MMP-9 protein by Western blot analysis in rat bone marrow,and the expression of MMP-9 with immunofluorescence MMP-9 tags vascular endothelial cell by confocal microscopy,MMP-9 positive microvascular count in each group and the vascular basement membrane degradation situation by electronic microscope.After blocking the pathway with STAT3 inhibitor SH-4-54,the above indicators were tested again for functional verification,and the inter-group detection indicators were compared and analyzed.Results(1)on day 30 th,the blood routine(RBC,HB,HCT)of the rats in the hypoxia groups were higher than that in the control group(P<0.05).(2)The MVD in the bone marrow was significantly higher and the basal vascular membrane was significantly degraded in the hypoxia groups than that in the control group(P<0.05).(3)The serum IL-6 level in the hypoxia groups increased obviously compared that in the control rats(P<0.05).The P-JAK2?P-STAT3 and MMP-9 were significantly increased in the hypoxia groups compared to those in the control group(P<0.05).(4)The MMP-9 concentration in the bone marrow in CH rats were consistent with the analysis of MVD and BM degradation;(5)STAT3 inhibitor decreased the expression of P-STAT3?MMP-9 compared to the CH and CH+Solvent group.The P-STAT3?MMP-9 expression in the CH+STAT3 inhibitor group were obviously higher than those in the control group(P<0.05).(6)STAT3 inhibitor treatment significantly decreased the CH-induced MVD and the BM degradation(P<0.05).However,the MVD levels in the CH+inhibitor group were higher than those in the control group(P<0.05).(7)Treatment with a STAT3 inhibitor significantly decreased the P-JAK2 in the CH+STAT3 inhibitor group compared that in the CH group(P<0.05).(8)Treatment with a STAT3 inhibitor decreased the levels of RBC,HB,HCT and IL-6 in the CH+inhibitor group respectively compared to that in the CH and CH+Solvent groups(P<0.05).(9)There were no differences in levels of P-JAK2,P-STAT3 and MMP-9 between the CH and CH+Solvent groups(P>0.05).(10)There was no difference in the serum IL-6 levels between CH and CH+Solvent groups(P>0.05).Conclusions 1.Under chronic hypoxia,microvascular proliferated and basal membrane degradated in bone marrow of rats.In addition,IL-6 and its downstream P-JAK2 and P-STAT3 and MMP-9 expression furtherly increased.2.Under chronic hypoxia,with the application of STAT3 inhibitor SH-4-54,the expression of P-STAT3 and MMP-9 decreased.3.Under chronic hypoxia,bone marrow microangiogenesis and basal membrane degradation in rats was involved in IL-6/JAK2/STAT3/MMP-9 pathway.Part II The research on the basal membrane degradation of microvessel in the bone marrow of rats under different time hypoxia are related to MMP-9 and COL4A1.Objective Hypoxia can cause damage of multiple systems and organs in the body which include the basement membrane of blood vessels.As an important component of basement membrane,the COL4A1 is one of the important members.MMP-9 level increased under hypoxia whose biological function is the degradation of IV type collagen fibers.Our previous study found that hypoxia demaged the vascular basement membrane,but the mechanism is still unclear.In this study,the expression of MMP-9 in rat bone marrow under different hypoxic conditions and the effect and changes of COL4A1 in blood vessel basement membrane in bone marrow were investigated by establishing animal models of hypoxia at different time.Methods Rat animal models were established,with male SD rats as control group,hypoxia 3-d group,hypoxia 7-d group and hypoxia 10-d group,each with 20 cases as subjects.Hypoxic models were prepared in the low-pressure oxygen chamber of the plateau medical research center,and were simulated at an altitude of 7000 m for 24h/ day.The research detect the level of MMP-9 m RNA by RT-PCR and the protein of MMP-9 and COL4A1 by Western blot in rat bone marrow and vascular basement membrane degradation situation by electron microscope with acute hypoxia at different time.Results(1)on day 3th,the red blood cell(RBC),HB and HCT increased compared to the control group(P<0.05).On day 7th,the RBC,HB,and HCT increased compared to the 3-day-group(P<0.05).On day 10 th,the RBC,HB,and HCT increased respectively compared to the 7-day group(P<0.05).(2)under the hypoxia condition,the levels of MMP-9 m RNA and protein in the hypoxia groups increased compared that in the control group(P<0.05).The level of MMP-9 increased in the 10-day group compared to that in the 3-day and 7-day groups.Furthermore,in the 7-day group the level of MMP-9 was higher compared that in the 3-day group(P<0.05).(3)COL4A1 levels in bone marrow samples from the hypoxia groups were lower than those in the control group(P<0.05).The expression of COL4A1 in the 7-day group was lower than that in the 3-day group(P<0.05),and that in the 10-day group was lower than that in the 7-day group(P<0.05).(4)with the extension of hypoxic time,the degradation of the basal membrane of blood vessels in the bone marrow of rats was gradually aggravated.The control group showed a thick and continuous BM,while in the hypoxia groups,the BM was uneven and thin with increased BM degradation.The BM thickness in the 3-day group was significantly higher than that in all other hypoxia groups,while the BM thickness in the 10-day group was significantly lower than that in all other groups.(5)In rat's bone marrow under hypoxic condition,the expression of COL4A1 was consistent with the severity of substrate degradation.The increase of MMP-9 level was consistent with the decrease of COL4A1 expression.Conclusions 1.Under hypoxic condition,the basal membrane of blood vessels degraded.2.Under hypoxia,the level of MMP-9 increased and the COL4A1 expression decreased in bone marrow of rats.3.Under hypoxic condition,MMP-9 and COL4A1 are related to the degradation of vascular basal membrane.