Protective Effect Of TanⅡA On Hypoxic Injury Of PC-12 Cells And Its Mechanism

High altitude cerebral edema refers to brain tissue damage caused by high altitude and low oxygen environment.It is a disease caused by increased water content,resulting in increased brain size and weight,and eventually brain dysfunction.High altitude cerebral edema is one of the most serious altitude sickness with rapid onset and high mortality,and there is no ideal treatment at present.Salvia miltiorrhiza is a Salvia miltiorrhiza Bge plant in the Labiaceae family.The dried root and rhizome have the effect of promoting blood circulation and removing blood stasis.TanⅡA is a representative of lipopolysaccharide of Salvia miltiorrhiza.It is a monomer component isolated from the root of salvia miltiorrhiza.It has anti-inflammatory,anti-apoptotic and anti-oxidation effects.We proposed that TanⅡA may have pharmacological activities in the prevention and treatment of altitude disease and brain edema at high altitude.Therefore,in this study,we evaluated the effect and mechanism of TanⅡA on anti-hypoxia injury of PC-12 cells,providing experimental basis for the research and development of new anti-altitude disease drugs based on TanⅡA.Experimental methods:1.PC-12 cells were exposed to 1%oxygen for 0,6,12,18 and 24 h in hypoxic environment.Cell morphology changes,cell survival rate,LDH overflow rate,ROS and MDA content,CAT and SOD activity were observed to determine the best hypoxic injury detection time point.2.The effects of different concentrations of TanⅡA(1×10^-7mol/L～1×10^-4mol/L)on the survival rate of PC-12cells in hypoxic environment were studied,and the degree of nuclear pyknosis was observed by immunofluorescence staining,and the optimal concentration of TanⅡA to protect cells from hypoxic injury was determined.3.Immunofluorescence staining was used to observe the morphology of primary cilia.Western blot and RT-PCR were used to detect the effects of TanⅡA on primary cilia-related proteins and genes.4.Calcium probe was used to detect calcium content,Western blot and RT-PCR were used to detect TRPV4 protein gene expression,and immunofluorescence staining was used to explore the relationship between primary cilia and TRPV4.The effects of TanⅡA on Nrf2/ARE signaling pathway through TRPV4 were investigated by detecting the activity and expression levels of ROS and antioxidant enzymes.5.Western blot and RT-PCR were used to detect PDH2 protein and gene to explore the effect of hypoxia on PHD2,immunofluorescence staining was used to explore the relationship between primary cilia and PHD2,and immunofluorescence was used to detect HIF-1αnuclear translocation.Western blot was used to detect the effects of TanⅡA on PHD2/HIF-1αpathway related proteins,mitochondrial membrane potential and apoptosis-related proteins,and to explore the protective mechanism of TanⅡA against apoptosis in PC-12 cells.The main experimental results were as follows:1.With the prolongation of hypoxia treatment time,the survival rate of PC-12 cells significantly decreased,the LDH overflow rate,ROS and MDA contents significantly increased,and the activities of CAT and SOD also significantly increased and reached the peak at 18 h,then began to decline;2.Different concentrations of TanⅡA can improve the survival rate of PC-12cells treated with hypoxia,especially 1×10^-5mol/L has the most obvious protective effect,and has obvious reverse effect on the nuclear pyknosis caused by hypoxia,so TanⅡA is used in the subsequent studies.3.TanⅡA can protect the primary cilia,prolong the primary cilia and increase the expression of IFT88 protein and gene;4.The high expression of TRPV4 localized in primary cilia can cause calcium overload and then lead to a large increase of ROS.TanⅡA can inhibit the high expression of TRPV4 and reduce calcium overload to eliminate ROS.5.TanⅡA treatment increased the expression of PHD2 localized in primary cilia and no nuclear translocation of HIF-1αoccurred,leading to a significant reduction in the expression of HIF-1αsignaling pathway related proteins.Mitochondrial damage was improved,the expression of pro-apoptotic proteins decreased,and the expression of anti-apoptotic proteins increased.The results above showed that:1.The hypoxic injury model of PC-12 cells was successfully established in this study,and the optimal detection time point of hypoxic injury index was screened.2.TanⅡA had a significant protective effect on hypoxic injury of PC-12 cells,and the optimal protective concentration was 1×10^-5mol/L.3.TanⅡA has protective effect on primary cilia;4.TanⅡA plays an anti-hypoxic role by regulating Nrf2/ARE-related signaling pathway through TRPV4 located in primary cilia;5 TanⅡA plays an anti-apoptotic role by regulating HIF-1αrelated signaling pathways through PHD2 localized in primary cilia.