Lactate Enhances Arc/arg3.1 Expression Through HCAR1-β-arrestin2 Pathway In Astrocytes

Lactic acid(2-Hydroxypropanoic acid)is a type of carboxylic acid containing hydroxyl groups that is converted into pyruvate by lactate dehydrogenase during the glycolysis of glucose taken up by cells.Lactic acid has isomers at the same time,l-lactic acid and d-lactic acid,L-lactic acid is the main component.Lactic acid is constantly produced during normal metabolism and exercise,but its concentration does not generally rise.In fact,lactic acid was once considered as a waste of anaerobic metabolism,but now it is known that lactic acid is continuously formed under aerobic conditions.In the brain,astrocytes are the main sites for glycolysis during glucose uptake,so astrocytes are also the main sites for lactic acid production in the brain.With the discovery that astrocytes in the brain and lactic acid produced in the brain supply energy to neurons,this discovery is also known as the astrocyte-neuron-lactate shuttle(ANLS)hypothesis.In addition,hydroxycarboxylic acid receptor 1(HCAR1)has also been found as a specific receptor for lactic acid.Therefore,lactic acid is now widely considered as a major energy source,mainly glycogen heterogeneous precursor and signal molecule Role,not just as waste of anaerobic metabolism.HCAR1(hydrocarboxylic acid receptor 1),also known as G protein-coupled receptor 81(GPR81),was first discovered in adipocytes to be activated by physiological concentrations of lactate and HCAR1 agonist 3,5-dihydroxybenzoate so that to reduce cAMP levels,it is also a specific receptor for lactic acid.Studies have found lactate receptors in the brain,which indicates that lactate,especially L-lactic acid,is a signaling substance in the brain in addition to being a metabolite and energy substrate,however,L-lactic acid signals are also important for brain function.The impact has not been thoroughly studied.The focus of my article is also to study the effect of L-lactic acid as a signal on astrocytes in the brain,and to investigate whether the signal effect of L-lactic acid on astrocytes can affect neurodegenerative diseases.Neurodegenerative disease is the gradual loss of neuron structure and function,which deteriorates over time,including the death of neurons and disorders of glial cell balance.Neurodegenerative diseases can be divided into acute neurodegenerative diseases and chronic neurodegenerative diseases.The former mainly includes stroke and brain injury;the latter mainly includes Parkinson’s disease,Alzheimer’s disease,depression,Huntington’s disease,amyotrophic lateral sclerosis,etc.Although the disease sites and causes of these diseases are different,degenerative lesions of nerve cells are their common feature.Among them,the dysfunction and reaction of astrocytes in glial cells are related to the occurrence and development of neurodegenerative diseases.Therefore,astrocytes are also an important part of the study of neurodegenerative diseases.Astrocytes are the most widely distributed type of cells in the brain,as well as the largest type of cells in glial cells.The complex and balanced two-way stability between neurons and glial cells is an important basic environment for the development of the central nervous system(CNS).Astrocytes have the function of secreting trophic factors to protect and support neurons,and play a vital physiological role in removing neurotransmitters such as glutamate through glutamate uptake and regulating K+in synaptic cleft.Glutamate transporter(EAAT)and aquaporin 4(AQP4)water channels are expressed on astrocyte structures.They also participate in physiological functions in the brain and affect the development and occurrence of neurodegenerative diseases(ND).In addition,astrocytes are the main sites for the uptake of glucose in the brain and glycolysis to produce L-lactic acid.In the earliest discovered adipocytes,its own lactic acid acts on HCAR1 in the adipocytes,which causes the down regulation of cAMP in the adipocytes and inhibits the breakdown of fat and inflammation.Therefore,as the most widely distributed type of cells in the brain,the signaling effect on HCAR1 in astrocytes which L-lactic acid produced by itself and external exchange of lactate into the blood-brain barrier through the lymphatic glial system acted had become the focus of experimental research in this paper.Protecting astrocytes and astrocytes’ support to neurons plays a vital role in improving neurodegenerative diseases.In recent years,with the discovery that lactic acid can be used as an intercellular signaling molecule involved in synaptic plasticity,even the peripheral administration of lactic acid can produce antidepressant pharmacological effects.Here,our research focuses on HCAR1 on the most widely distributed astrocytes in the brain,and found and verified that lactic acid can cause the overexpression of Arc,a cell-activation protein in astrocytes,through HCAR1.However,the expression of Arc does not depend on the GI protein pathway of HCAR1.We found that lactic acid enhances the expression of Arc protein through the HCARl-β-arrestin2 pathway.In summary,lactic acid acts HCAR1 on astrocytes,and enhances the expression of MAPK-dependent Arc through β-arrestin2,this reduces the influx of calcium ions when astrocytes are exposed to glutamate damage,and thus protects astrocytes,which may indirectly enhance the uptake of glutamic acid by astrocytes,which also implies HCAR1 is a potential target for improving neurodegenerative diseases caused by glutamate damage.OBJECTIVE:To study the mechanism by which lactic acid enhances the expression of Arc/arg3.1 in astrocytes.METHODS:C57BL/6J newborn mice and β-arrestin2 knockout newborn mice about 3 days after birth were used to extract primary astrocytes.The primary astrocytes were cultured in vitro,and after two passages,the astrocyte-activated marker glial fibrillary acidic protein(GFAP)was used to detect astrocytes with a positive rate greater than 95%.This experimental process mainly used L-lactic acid stimulation to detect related indicators in the control group and the experimental group.1.The distribution of HCAR1 in the brain group was detected by immunohistochemistry using brain slices from wild-type mice,and the presence of HCAR1 in astrocytes was verified by immunofluorescence from mouse hippocampal tissue samples.2.CCK-8 test was used to detect the optimal concentration of L-lactic acid for astrocytes at physiological concentration of 0-20mM.3.After stimulating primary astrocytes with the optimal concentration of L-lactic acid,use RT-QPCR to detect the expression of relevant factors provided in the literature,so as to determine the significant effect of L-lactic acid on astrocytes.4.We obtained the HCAR1 gene knockout U87 cell line through CRISPR-Cas9 and stimulated the cell line with L-lactic acid,and used SiRNA to interfere with the expression of HCAR1 in primary astrocytes,and then given L-lactic acid stimulation Finally,Western Blotting was used to detect whether Arc expression was related to HCAR1.5.In vitro cultured primary astrocytes,β-arrestinl knockout,β-arrestin2 knockout,and cells transfected with β-arrestin2 overexpression plasmid were stimulated with L-lactic acid respectively,and whether expression of Arc related to GI protein and β-arrestin was detected by Western Blotting.6.Using MAPK inhibitors PD980590 and SB203580 on astrocytes to verify that the expression of ARC under L-lactic acid stimulation is related to MAPK.7.After astrocytes and HCAR1 knockout cell lines and β-arrestin2 knockout astrocytes were stimulated by glutamate damage,the experiment is divided into the following groups including primary astrocytes in control group,control+L-lactic acid administration group,HCAR1 interference group,HCAR1 interference group+L-lactic acid administration group,β-arrestin2 knockout group,β-arrestin2 knockout+L-lactic acid administration group.Finally,the changes of calcium ion influx in primary astrocytes in the drug group were tested by flow cytometry.Then verified the protective effect of L-lactic acid on astrocytes under glutamate damage through AV/PI experiments.HCAR1 interference group,HCAR1+L-lactic acid administration group,β-arrestin2 knockout group,β-arrestin2 knockout+L-lactic acid administration group.RESULTS1.HCAR1 widely exists in the brain and HCAR1 was expressed in astrocytes.Immunohistochemical analysis of tissue sections from mouse brain regions(cortex,corpus callosum,hippocampus,cerebellum,choroid)showed that HCAR1 is widely expressed in the brain,and the presence of HCAR1 in astrocytes was confirmed by immunofluorescence2.Lactic acid causes significant upregulation of Arc/arg3.1 in many trophic factors in astrocytes CCK-8 experiments verified that the optimal concentration of L-lactic acid for astrocytes was 0.5mM,and the results of RT-QPCR experiments found that L-lactic acid significantly increased the expression of Arc and arg3.1(P<0.0001)Increased expression of Arc caused by lactic acid was also verified by Western Blotting experiments3.Lactic acid causes upregulation of Arc protein expression through HCAR1 After L-lactic acid stimulation in HCAR1 knockout cell lines and primary astrocytes transfected with HCAR1 siRNA,protein expression of ARC no longer increased,suggesting that lactic acid causes upregulation of Arc protein through HCAR1.4.Lactic acid mediated upregulation of Arc/arg3.1 through β-arrestin2,but not through the Gi protein pathway of HCAR1.After stimulation with L-lactic acid,the expression of ARC protein in primary astrocytes knocked out by β-arrestin2 no longer increased,and the expression of ARC protein in primary astrocytes knocked out by β-arrestinl still increased.And the use of pertussis toxin PTX did not affect the expression of ARC protein in astrocytes,suggesting that lactic acid may not mediate up-regulation of Arc/arg3.1 expression through the Gi protein pathway in HCAR1,it is through HCAR1/β-arrestin2 pathway.And after over-expression of β-arrestin2,Arc protein expression was more significant.5.Lactic acid enhances MAPK-dependent Arc/arg3.1 expression through the β-arrestin2-MAPK pathwayAfter using MAPK inhibitors PD980590 and SB230580 on astrocytes,the expression of Arc no longer increased under L-lactic acid stimulation.6.Lactic acid up-regulates Arc through the HCAR1-β-arrestin2 pathway to reduce calcium influx and apoptosis in astrocytes caused by glutamate damage.In the case of glutamic acid damage,cell flow cytometry was used to detect the concentration of calcium ion probe flour-3 AM under the stimulation of L-lactic acid to reflect the change of calcium influx.The results show that lactic acid can reduce calcium ion in astrocyte Influx was eliminated after HCAR1 interference and β-arrestin2 knockout.The results of AV/PI experiments proved that L-lactic acid reduced astrocyte apoptosis in the case of glutamate damage,and this phenomenon was also cancelled after HCAR1 interference and β-arrestin2 knockout.CONCLUSION1.Lactic acid enhances Arc/arg3.1 expression through the HCAR1-β-arrestin2 pathway2.L-lactic acid can improve astrocyte damage under glutamate damage and reduce apoptosis under glutamate damage.In summary,this experiment has the following innovations:1.It was found that lactic acid is a signal molecule that enhances the expression of Arc protein in astrocytes,which can reduce the damage of astrocytes in the face of glutamic acid and has a certain protective effect on astrocytes.2.To propose that HCAR1 is an important target for the improvement and treatment of neurodegenerative diseases caused by glutamate.