| Objective:Circadian rhythm is a biological rhythm established by the diurnal program encoded by genes.It controls the physiological activities of many organisms and is an important adaptation mechanism to the periodic changes of light/dark on the earth.The circadian rhythm system and the core circadian rhythm proteins Bmal1 and Per2 also play an essential role in aging.Studies have shown that mice with Bmal1 and Per2 knockout demonstrated the characteristics of premature aging.Aging is a complex process,which is affected by exogenous and endogenous factors,and affects physiological functions through the loss of cellular functions.Aging also affects the circadian rhythm of the organism.The periodic expression and amplitude of the circadian rhythm change in the elderly,and tissue and cell synchronization disorder correspondingly.In modern society,stress and irregular lifestyle(exposure to light at night)will cause circadian rhythm disorders,and accelerate the process of aging,resulting in a series of dysfunctions.In this study,a mouse aging model with circadian rhythm disturbance was established by Dgalactose(D-gal)combined with continuous light exposure to explore the underlying mechanisms and anti-brain aging effect of Chinese traditional medicine polygala tenuifolia by regulating circadian rhythm.Methods:1.The germline mice were divided into control group(NC),model group(Dgal+light),and Polygala tenuifolia group(PT).In addition to the NC group,the mice in the D-gal+light group and PT group were continuously injected subcutaneously with D-gal(1000mg/Kg)for 60 days,and received continuous light(1000lux)during the last 30 days.At the same time,the PT group was treated with Polygala tenuifolia extract(300mg/Kg)by gavage,and D-gal+light group was given equal normal saline.The mice in NC group were not exposed to light,and the same amount of normal saline was injected subcutaneously and gavage.2.After administration,cognitive function was analyzed by using Morris water maze test.3.Real-time PCR was used to detect the m RNA levels of Bmal1 and Per2 at each circadian time point(CT).4.The m RNA levels and protein expressions of P21 and P16 in hippocampus were detect by Real-time PCR and Western Blot.At the same time,β-galactosidase staining was used to evaluated the senescence state of hippocampal cells.5.Immunohistochemistry was used to detected the expression of synapseassociated protein Ng and SYT-1 in hippocampus.6.Network pharmacology was used to analyze the targets of Polygala tenuifolia and disease brain aging.KEGG was used to enrichment the pathway of intersection target.The signal pathway related to circadian rhythm and brain aging was analyzed.7.According to the results of network pharmacology analysis,the key proteins in related pathways were detected by Western Blot,and the calcium content in hippocampus was detected.Endoplasmic reticulum stress-related proteins and pm TOR/m TOR protein were also detected.8.Real-time PCR,Western Blot and immunohistochemistry were used to detect the m RNA and protein expression levels of autophagy-related proteins LC3,Beclin1 and P62 in hippocampus.The expressions of apoptosis-related proteins caspase3 and caspase12 in hippocampus were detected by Western Blot.9.The content of senegenin in the Polygala tenuifolia extract was analyzed by liquid chromatography.10.CCK8 method was used to screen the optimal concentration of each drug(200 m M for carbachol,60μM for senegenin,and 50μM for 2-APB).Nerve cells HT22 were divided into 4 groups: control group(NC),200μM IP3 R agonist carbachol group(CAR),60μM senegenin group(SEN),and 60μM senegenin +50μM IP3 R inhibitor 2-APB group(SEN+2-APB).11.The Real-time PCR detection of each cell biological clock gene Bmal1,Per2 and aging markers P21,P16 m RNA level.The cell senescence of each group was detected by β-galactosidase staining.12.Western Blot was used to detect the expression of circadian rhythm-related signaling pathway proteins IP3 R,Ca MKⅡ,ERK,CREB and endoplasmic reticulum stress-related proteins p-perk,GRP78,ATF4 in each group.13.Fluo-4 fluorescence probe method is used to detect the HT22 the intracellular calcium ion concentration.14.The expressions of autophagy-related proteins LC3 and P62 in HT22 cells was detected by immunofluorescence method.Results:1.In the 5-day location navigation test,the escape latency of the three groups of mice was statistically different since the third day.Compared with NC group,the escape latency of mice in D-gal+light group was significantly prolonged(p<0.01,p<0.01,p<0.01),while the escape latency of mice in PT group was significantly shortened(p<0.01,p<0.01,p<0.01).In the space exploration test,the number of shuttling to the hidden platform and the time of staying in the target quadrant in the D-gal+light group were lower than those in the NC group(p<0.01,p<0.001).However,compared with the D-gal+light group,the number of shuttling to the hidden platform and the time of staying in the target quadrant of the PT group increased(p<0.01,p<0.01).There were no significant differences among the three groups in the platform elevation and swimming speed tests.2.Compared with the NC group,the expression of circadian clock genes Bmal1 and Per2 in the hippocampus of mice in the D-gal+light group was decreased at CT6,CT12,CT18 and CT24,and the rhythm disappeared.Compared with the D-gal+light group,the PT group recovered the expression of clock genes Bmal1 and Per2 at each time point and restored the rhythm to a certain extent.3.For cellular senescence in the hippocampus of mice,compared with the NC group,the m RNA levels of aging markers P21 and P16 were increased in Dgal+light group(p<0.001,p<0.05),and the m RNA levels of P21 and P16 in PT group significantly were decreased compared with D-gal+light group(p<0.001,p<0.01).The results of protein detection also showed that the expression of P21 and P16 in the D-gal+light group was higher than that in the NC group(p<0.05,p<0.05),and the expression of PT group was lower than that in the D-gal+light group(p<0.01,p<0.01).The results of β-galactosidase staining showed that compared with the NC group,the expression of β-galactosidase in the D-gal+light group was increased(p<0.001,p<0.001),and the expression of β-galactosidase in the PT group was decreased compared with the D-gal+light group(p<0.001,p<0.001).4.Compared with NC group,D-gal + light group mice hippocampus Ng and SYT-1 expression was decreased obviously(p<0.001,p<0.001),while PT group was significantly increased compared with D-gal+light group(p<0.01,p<0.01).5.Network pharmacological analysis showed that Polygala Tenuifolia Extract regulates circadian rhythm involving IP3R/Ca MKⅡ/ERK/CREB signaling pathway.Compared with the NC group,the expression of IP3 R in the D-gal+light group was increased(p<0.01),and the expression of p-Ca MKⅡ,p-ERK and pCREB proteins was decreased(p<0.01,p<0.001,p<0.01).Compared with Dgal+light group,IP3 R was decreased(p<0.05),and the protein expressions of pCa MKⅡ,p-ERK and p-CREB were increased in PT group(p<0.05,p<0.01,p<0.05).The content of calcium in the hippocampus of mice in the D-gal+light group was increased compared with the NC group(p<0.5),and it in the PT group was decreased compared with the D-gal+light group(p<0.5).6.Compared with NC group,the expressions of p-perk,GRP78,ATF4 and pm TOR in D-gal+light group were increased(p<0.05,p<0.01,p<0.01,p<0.01).Compared with D-gal+light group,p-perk,GRP78,ATF4 and p-m TOR expression decreased in PT group(p<0.05,p<0.05,p<0.05,p<0.01).7.Compared with the NC group,m RNA levels of autophagy related genes LC3,Beclin1 and P62 were significantly increased in the D-gal+light group(p<0.001,p<0.001,p<0.01).Compared with the D-gal+light group,the m RNA level in the PT group was decreased(p<0.01,p<0.001,p<0.001).The protein expressions of Beclin1,LC3 and P62 in each group were consistent with the m RNA results.Immunohistochemical results of LC3 and P62 showed that the expressions of autophagy related proteins LC3 and P62 in D-gal+light group were increased compared with NC group(p<0.01,p<0.001),and the protein expressions in PT group were decreased compared with D-gal+light group(p<0.01,p<0.001).8.Compared with the NC group,the expression of caspase12 and caspase3 in the D-gal+light group was increased(p<0.05,p<0.01).Compared with the Dgal+light group,the expression of caspase12 and caspase3 in the PT group was decreased(p<0.05,p<0.01).9.The results of liquid chromatography showed that the Polygala Tenuifolia Extract contained 0.12mg/ml of senegenin.10.In vitro,compared with the NC group,the m RNA levels of clock genes Bmal1 and Per2 were decreased in the CAR group(p<0.001,p<0.05).The m RNA levels of Bmal1 and Per2 in the SEN group(p<0.05,p<0.001)and the SEN+2-APB group(p<0.001,p<0.001)were increased,compared with CAR group.And the increase in the SEN+2-APB group was more significant than that in the SEN group(p<0.05,p<0.05).Meanwhile,m RNA levels of aging genes P21 and P16 in CAR group were higher than those in NC group(p<0.001,p<0.01).Compared with the CAR group,the SEN group(p<0.001,p<0.001)and SEN+2-APB group(p<0.001,p<0.001)significantly decreased,and the SEN+2-APB group decreased more significantly than the SEN group(p<0.05,p<0.05).The results of β-galactosidase staining showed that compared with the NC group,theβ-galactosidase staining positive cells in the CAR group was significantly increased(p<0.001),and the β-galactosidase staining positive cells in the SEN group and SEN+2-APB group was significantly lower than that in the CAR group(p<0.01,p<0.001),and the SEN+2-APB group was the lowest.11.Compared with NC group,IP3 R protein expression in CAR group was increased(p<0.05),and p-Ca MKⅡ,p-ERK,p-CREB protein expression was decreased(p< 0.01,p<0.01,p<0.01).Compared with CAR group,the expression of IP3 R was decreased in the SEN group(p<0.01)and the SEN+2-APB group(p<0.01),and the protein expressions of p-Ca MKⅡ,p-ERK,p-CREB were increased in SEN group(p<0.05,p<0.05,p<0.05)and SEN+2-APB group(p<0.01,p<0.001,p<0.001).There were also significant differences between SEN+2-APB group and SEN group.12.The expression of endoplasmic reticulum stress-related proteins p-perk,GRP78 and ATF4 in the CAR group was higher than that in the NC group(p<0.05,p<0.01,p<0.05).The results in SEN group(p<0.01,p<0.01,p<0.05)and 2-APB+SEN group(p<0.001,p<0.01,p<0.01)were lower than those in CAR group.The protein expression in SEN+2-APB group was also significantly different from that in SEN group(p<0.05,p<0.05,p<0.05).13.The results of intracellular calcium concentration showed that: Compared with the NC group,the intracellular calcium concentration in the CAR group was increased(p<0.001),the calcium concentration in the SEN group(p<0.001)and SEN+2-APB group(p<0.01)was decreased compared with the CAR group.And the calcium concentration in the SEN+2-APB group was lower than that in the SEN group(p<0.05).14.Compared with the NC group,LC3 and P62 expression in the CAR group was increased(p<0.001,p<0.001);and the expression of LC3 and P62 in the SEN group(p<0.001,p<0.001)and the SEN+2-APB group(p<0.001,p<0.001)was significantly lower than the CAR group.The expressions of LC3 and P62 were lower in SEN+2-APB group.Conclusion1.D-galactose combined with continuous light exposure caused the circadian rhythm disorder in mice and accelerated the brain aging and cognitive disfunction.Polygala tenuifolia extract can delay aging and improve cognitive function in mice by restoring the disordered circadian rhythm.2.Polygala tenuifolia extract can regulate the expression of IP3 R,which activates CamkⅡ/ERK/CREB signaling pathway,on the other hand,inhibits endoplasmic reticulum stress to restore the rhythm of circadian regulation factors and disordered circadian rhythm,thereby enhancing autophagy and reducing apoptosis,consequently plays a role in delaying brain aging.This effect may be associated with senigenin,the main component of Polygala tenuifolia extract. |
PDF Full Text Request