Effect Of Cell Lipid Raft On Leptospira Infection And Its Potential Therapeutic Value

Leptospirosis is a zoonotic infectious disease caused by pathogenic leptospirosis,which spreads widely around the world,and is a natural infectious disease caused by the mutual source of human and animal.Infected people or animals experience jaundice,miscarriage,sepsis,liver and kidney failure,and even death.Leptospira colonizes the host's kidneys for a long time after infection and is excreted in urine.It can survive for several months in a humid environment and remain infectious,posing a serious threat to public health safety and the development of animal husbandry.Therefore,revealing the internalization mechanism of leptospira can help to control its infection from the source and reduce its damage to the body and excretion of leptospira urine.However,there are few studies on the mechanism of leptospira entry.Cellular lipid rafts are nanoscale membrane domains in lipid membranes,which are rich in cholesterol and spphingolipids and contain a variety of signal transduction proteins and transporters.It has been reported that more and more pathogens require the participation of lipid rafts in the process of internalization.In this study,the relationship between leptospiral internalization and lipid raft was studied to provide a theoretical basis for clinical screening of effective drugs for leptospirosis.Firstly,in vitro experiments were conducted to verify whether lipid raft was related to leptospiral internalization.The lipid raft structure of J774 A.1 cells was disrupted by M?CD,and the intracellular leptospiral burden was significantly reduced by q PCR detection.After cholesterol restored the lipid raft structure,the burden of intracellular leptospires increased.At the same time,the Cav-1 si RNA was used to disrupt J774 A.1 cell lipid raft structure,the burden of intracellular leptosipres is also significantly reduced.The above results proved that leptospira requires the integrity of lipid rafts to enter the cell.In addition,the same experiment was performed on Vero cells,and the results also showed that the leptospira entered the cell dependent on lipid rafts,and proved that the leptospira entry into the cell required lipid rafts and had nothing to do with the phagocytosis of macrophages.The transmission electron microscope observation of the intracellular leptospires showed that the burden of intracellular leptospires in the lipid raft disruption group of J774 A.1 cells was significantly reduced at 40 min and 4 h after infection,and the morphology of leptospira changed,indicating after the lipid rafts disruption,the ability of cells to eliminate leptospira was enhanced.q PCR and leptospira culture count also proved that the burden of intracellular leptospires were basically killed in the lipid raft disruption group 4 h after infection.Western Blot results showed that after lipid rafts disruption,autophagy was activated.In order to explore whether the enhancement of the ability of cells to remove leptospira is related to autophagy,cells were stimulated with rapamycin and 3MA respectively.The results of leptospira culture count showed that the burden of leptospires in the rapamycin-treated group was significantly reduced,indicating that the increase in the level of autophagy helps to kill leptospira.The above research work showed that leptospira can enter the cell through lipid rafts to avoid activating autophagy.We used M?CD and dipotassium glycyrrhizinate(a triterpene sugar isolated from licorice root,which has the biological function of destroying the integrity of cell lipid rafts)in vivo and in vitro experiments to explore whether drugs that interfere with cell lipid rafts have the effect of resisting leptospira infection.In vitro experiments showed that dipotassium glycyrrhizinate could prevent leptospira from entering cells after 2 h pretreatment.Different animal models showed that M?CD and dipotassium glycyrrhizinate could not improve the survival rate of golden hamsters after leptospiral infection,which proved that M?CD and dipotassium glycyrrhizinate could not treat acute leptospirosis.However,in the chronic leptospirosis model,M?CD and dipotassium glycyrrhizinate significantly alleviated the pathological damage caused by leptospiral infection and reduced leptospiral colonization in the kidney.In addition,dipotassium glycyrrhizinate can reduce the release of inflammatory cytokines in kidney tissue.In the study of the mechanism of dipotassium glycyrrhizinate,it has been found that dipotassium glycyrrhizinate can reduce the cytokines(IL-1?,IL-6,TNF-?),chemokines(RANTES,MCP-1)and i NOS m RNA expression of TCMK-1 cells caused by leptospiral infection,reduce the secretion of cytokines(IL-1?,TNF-?),and inhibit the activation of NF-?B and MAPK signaling pathways.In summary,this experiment proved that leptospira requires lipid rafts to enter cells.After the lipid rafts are disrupted,the number of leptospires entering cells is significantly reduced,and the ability of cells to kill leptospires is enhanced,which is related to the increase in the level of autophagy.The application of drugs M?CD and dipotassium glycyrrhizinate that interfere with cell lipid rafts is not effective for acute leptospirosis,but can treat chronic leptospirosis.