Tu-Xian Decoction Protective Cognitive Impairment In Diabetic Rats By Death-associated Protein Kinase 1

[Objective]Diabetic Cognitive Impairment(DCI)is a chronic complication of diabetes,and its exact pathogenesis is still unclear.In DCI,the syndrome of "kidney deficiency and blood stasis,unhealthy brain" is the most common.Tu-Xian decoction(TXD)has the function of "tonifying kidney and blood circulation,benefiting brain and strengthening intelligence".Death-associated protein kinase 1(DAPK-1)is involved in various neurodegenerative diseases.Through metabolomics,in vitro culture and animal experiments,this topic explores whether Tu-Xian decoction can protect cognitive impairment in diabetic rats by inhibiting DAPK-1.[Methods]Part 1The main components of Tu-Xian decoction were identified by Ultra highperformance liquid chromatography-mass spectrometry(UHPLC-MS)non-target metabolomics technology.Search the literature to check whether the main components in TXD have the function of regulating DAPK-1.Part 2The primary hippocampal neurons were isolated from newborn 24-hour SD rats,purified by lymphocyte separation medium density gradient centrifugation and differential adhesion method,and the neurons were divided into 4 groups:control group(Con,glucose content 25mmol/L),high glucose group(HG,glucose content 50mmol/L),Tu-Xian decoction group(TXD,glucose content 50mmol/L,set 0.1mg/mL,1.Omg/mL,5.0mg/mL,10.0 mg/mL,50.0mg/mL 5 TXD concentration gradients),DAPK-1 inhibitor group(TC-DAPK6,glucose content 50mmol/L,set 0.1 μmol/L,1.0μmol/L,10.0μmol/L,20.0μmol/L 4 concentration gradients of TC-DAPK6),the neurons were intervened in groups for 72 hours,and the relative activity of hippocampal neurons was measured by CCK-8 method.Divide the neurons into 3 groups:control group(Con),high glucose group(HG),and Tu-Xian decoction group(TXD,using the concentration of the group with the highest relative activity of neurons in the above CCK-8 test,that is,the optimal concentration intervention),after 72 hours of group intervention,the expression levels of T-DAPK-1 and P-DAPK-1(Ser308)were determined by Western blotting.Part 3The diabetic rat model was established by high-sugar high-fat(HSHF)diet combined with 35 mg/Kg streptozotocin(STZ).Diabetic rats were randomly divided into:Diabetic(DM)group,DAPK-1 inhibitor(TC-DAPK6)group,high-dose Tu-Xian decoction(H-TXD,12g/Kg)group,medium-dose Tu-Xian decoction(M-TXD,6g/Kg)group,low-dose Tu-Xian decoction(L-TXD,3g/Kg)group.Rats in the control(Con)group were fed with normal diet.After 12 weeks of group intervention,the cognitive function of the rats was detected by Morris water maze,the volume of the hippocampus of the rats was detected by cranial magnetic resonance imaging,the pathological changes of the brain were detected by hematoxylin and eosin staining and Nissl staining,and Aβ,APP,P-APP,Tau,P-Tau(Thr231),P-Tau(Ser396),Caspase-3,T-DAPK-1,P-DAPK-1(Ser308)protein expression levels in the hippocampus of rats was detected by Western blotting.The expression level of Bcl-2 in rat hippocampus was detected by immunohistochemistry.[Results]Part 1TXD was identified to contain 908 known compounds including flavonoids,phenols,terpenoids,phenylpropanes,alkaloids,aliphatic acylates,organic acids and their derivatives,sugars and their derivatives.Part 21.The primary hippocampal neurons were isolated,and the purity was over 95%as identified by neuron-specific enolase(NSE)immunofluorescence.2.After 72 hours of group intervention,the relative activity of neurons in the HG group was(79.23±5.62)%/Con,and the relative activity of neurons in each dose of TuXian decoction group was:0.1mg/mL TXD group(79.83±4.02)%/Con;1.Omg/mL TXD group(100.60±7.59)%/Con;5.0mg/mL TXD group(116.03±6.99)%/Con;10.0mg/mL TXD group(93.53±2.78)%/Con;50.0mg/mL TXD group(92.17±5.07)%/Con.Compared with the Con group,the relative activity of neurons in the HG group decreased(P<0.01).Compared with the HG group,the relative activity of neurons in the 1.0mg/mL TXD group(P<0.01)and 5.0mg/mLTXD group(P<0.0001)increased.After 72 hours of group intervention,the relative activity of neurons in each dose of DAPK-1 inhibitor group was:0.1μmol/L TC-DAPK6 group(87.23±3.08)%/Con;1.0μmol/L TC-DAPK6 group(92.08±2.81)%/Con;10.0μmol/L TC-DAPK6 group(102.33±7.75)%/Con;20.0μmol/L TC-DAPK6 group(85.80±6.35)%/Con.Compared with the HG group,the relative activity of neurons in the 10.0μmol/L TC-DAPK6 group increased(P<0.01).3.Compared with Con group((1.16±0.14)/β actin),the expression level of TDAPK-1 in neurons of HG group((1.97±0.14)/β actin)increased(P<0.05).Compared with Con group((0.57±0.10)/T-DAPK-1),the expression level of P-DAPK-1(Ser308)in neurons of HG group((0.29±0.07)/T-DAPK-1)decreased(P<0.05),indicating that DAPK-1 activity increased.Compared with the HG group,there was no significant difference in the expression level of T-DAPK-1((1.83±0.16)/β actin)in the 5mg/mL TXD group(P>0.05),but P-DAPK-1(Ser308)((0.48±0.09)/T-DAPK-1)expression level in the 5mg/mL TXD group increased(P<0.05),indicating that DAPK-1 activity decreased.Part 31.Successfully established a diabetic rat model.The behavioral characteristics of cognitive decline in diabetic rats were found by the Morris water maze test.In the hidden platform experiment,compared with the Con group(Day 3:20.8±17.2s;Day 4:12.9±6.0s;Day 5:12.2±4.6s),the escape latency of the DM group on Days 3-5(Day 3:40.8±20.0,P<0.05s;Day 4:35.1 ±20.6s,P<0.0001;Day 5:28.1 ±21.0s,P<0.05)increased;compared with Con group(Day 3:461.4±493.9cm;Day 4:234.9±172.2cm;Day 5:223.1±141.6cm),the escape distance of the DM group on Days 3-5(Day 3:936.4±571.1cm,P<0.05;Day 4:852.6±629.8cm,P<0.0001;Day 5:686.5±634.9cm,P<0.01)increased.In the probe trial,compared with the Con group(3.25±0.71 times),the number of times of rats in the DM group crossed the target platform(1.17±0.41 times)decreased(P<0.05);Compared with Con group(28.7±3.6s),rats in DM group spent less time(17.9±3.9s)in the target platform quadrant(P<0.0001).2.The Morris water maze was used to detect the effects of TXD and DAPK-1 inhibitor(TC-DAPK6)intervention on spatial learning and memory abilities of diabetic rats.In the hidden platform experiment,compared with the DM group(Day 3:40.8±20.0s;Day 4:35.1 ±20.6s),escape latency of H-TXD group on Day 3(25.7± 19.1s),TC-DAPK6 group on Day 3(25.6± 18.7s),TC-DAPK6 group on Day 4(18.9± 10.9s)decreased(P<0.05);compared with the DM group(Day 3:936.4±571.1m;Day 4:852.6±629.8cm),the escape latency of H-TXD group on Day 3(518.7±466.4cm),HTXD group on Day 4(436.3±369.4cm),TC-DAPK6 group on Day 4(399.6±287.6cm)decreased(P<0.05).There were no significant differences in escape latency and escape distance among H-TXD group,M-TXD group,L-TXD group,and TC-DAPK6 group(P>0.05).In the probe trial,compared with the DM group(1.17±0.41 times),the times of crossing the target platform of the H-TXD group(3.17±0.75 times,P<0.0001),the MTXD group(2.57±0.79 times,P<0.01)and the TC-DAPK6 group(2.67±0.52 times,P<0.01)increased;compared with the DM group(17.9±3.9s),rats in the H-TXD group(25.1±3.3s,P<0.01),the M-TXD group(23.4±2.6s,P<0.05)and the TC-DAPK6 group(23.2±1.3s,P<0.05)spent longer in the quadrant of the target platform.There were no significant differences in the number of crossing the target platform and the time spent in the quadrant of the target platform among H-TXD group,M-TXD group,L-TXD group,and TC-DAPK6 group(P>0.05).3.Brain magnetic resonance imaging was used to evaluate the effect of TXD and DAPK-1 inhibitor(TC-DAPK6)intervention on the hippocampal volume of diabetic rats:compared with the Con group(147.13±5.24mm2),the total hippocampus volume of diabetic rats in the DM group(119.62±8.11mm2)decreased(P<0.05).Compared with the DM group,H-TXD,M-TXD,L-TXD,and TC-DAPK6 intervention had no statistically significant changes in hippocampus volume(P>0.05).4.Hematoxylin and eosin staining,Nissl staining to detect TXD,DAPK-1 inhibitor(TC-DAPK6)intervention in the pathological changes of the hippocampus of diabetic rats:compared with the Con group,the pathological sections of the rats in the DM group showed the distribution of neurons in the hippocampus,layer incompleteness,apoptosis,hypertrophic astrocytes,and granular degeneration.Compared with the DM group,the pathological sections of the brains of rats in each dose of TXD group and TC-DAPK6 group showed that the structure of the hippocampus were more complete,the layers were clearer,and the abnormal cells were less.Compared with the Con group(39.7±3.2/FOV),the number of Nissl bodies in the CA1 area of rats in the DM group(15.3±5.5/FOV)decreased(P<0.001).Compared with the DM group,the number of Nissl bodies in the CA1 area of rats in the H-TXD group(33.0±6.11FOV,P<0.01),the M-TXD group(29.7±2.1/FOV,P<0.05),the L-TXD group(31.3±3.1/FOV,P<0.01),TC-DAPK6 group(30.0±4.0/FOV,P<0.05)increased.5.Western blotting to detect the effect of TXD and DAPK-1 inhibitor(TC-DAPK6)on protein expression in hippocampus of diabetic rats.Compared with the Con group(Aβ:0.14±0.05/β actin),the expression level of Aβ(0.53±0.15/β actin)in the hippocampus of rats in the DM group increased(P<0.01).Compared with the DM group,there were no significant differences in the expression level of Aβ in the hippocampus of rats in the H-TXD group,M-TXD group,L-TXD group,and TC-DAPK6 group(P>0.05);There were no significant differences in the expression level of hippocampal Aβ among rats in the H-TXD group,M-TXD group,LTXD group,and TC-DAPK6 group(P>0.05).Compared with the Con group(P-APP:0.15±0.05/APP),the expression level of PAPP(0.81 ±0.11/APP)in the hippocampus of rats in the DM group increased(P<0.001).Compared with DM group,the expression level of P-APP in hippocampus of rats in HTXD group(0.24±0.07/APP,P<0.001)and TC-DAPK6 group(0.35±0.08/APP,P<0.01)decreased.Compared with the H-TXD group,the expression levels of P-APP(Thr668)in the hippocampus of rats in the M-TXD(0.55±0.17/APP)and L-TXD(0.57±0.19/APP)groups increased(P<0.05),while the expression levels of P-APP(Thr668)of rats in the TC-DAPK6 group were not significantly different(P>0.05).Compared with the Con group(P-Tau(Thr231):0.44±0.24/Tau;P-Tau(Ser396):0.27±0.15/Tau),the hippocampal P-Tau(Thr231)(1.42±0.46/Tau,P<0.01),P-Tau(Ser396)(1.66±0.37/Tau,P<0.0001)of the DM group rats increased.Compared with the DM group,the expression level of P-Tau in the hippocampus of the H-TXD group(P-Tau(Thr231):0.51±0.10/Tau,P<0.01;P-Tau(Ser396):0.2110.07/Tau,P<0.0001)± the MTXD group(P-Tau(Thr231):0.69±0.15/Tau,P<0.05;P-Tau(Ser396):0.46±0.25/Tau,P<0.0001),L-TXD group(P-Tau(Thr231):0.74±0.13/Tau,P<0.05;P-Tau(Ser396):0.52±0.18/Tau,P<0.001),TC-DAPK6 group(P-Tau(Thr231):0.41±0.18/Tau,P<0.01;P-Tau(Ser396):0.33±0.22/Tau,P<0.001)decreased.There were no significant differences in the expression level of P-Tau in hippocampus among rats in H-TXD group,M-TXD group,L-TXD group,and TC-DAPK6 group(P>0.05).Compared with the Con group(0.50±0.15/β actin),the expression level of Caspase3(1.14±0.27/β actin)in the hippocampus of diabetic rats in the DM group increased(P<0.01).Compared with the DM group,the hippocampal Caspase-3 expression levels were decreased in the H-TXD(0.41±0.10/β actin,P<0.001),M-TXD(0.51±0.11/p actin,P<0.01),L-TXD(0.45±0.11/β actin,P<0.01)and TC-DAPK6(0.44±0.09/β actin,P<0.01)groups of rats.There was no significant difference in the expression level of Caspase-3 in rat hippocampus among H-TXD group,M-TXD group,L-TXD group,and TC-DAPK6 group(P>0.05).Compared with the Con group(0.55±0.14/β actin),the expression level of T-DAPK1(1.14±0.31/β actin)in the hippocampus of rats in the DM group increased(P<0.05).Compared with the DM group,there were no significant differences in the expression level of T-DAPK-1 in the hippocampus of rats in the H-TXD group,M-TXD group,LTXD group,and TC-DAPK6 group(P>0.05).There were no significant differences in the expression level of T-DAPK-1 in rat hippocampus among H-TXD group,M-TXD group,L-TXD group and TC-DAPK6 group(P>0.05).Compared with the Con group(0.80±0.18/T-DAPK-1),the expression level of PDAPK-1(Ser308)(0.14±0.05/T-DAPK-1)in the hippocampus of rats in the DM group decreased(P<0.05),indicating that increased DAPK-1 activity.Compared with DM group,the expression level of P-DAPK-1(Ser308)in the hippocampus of rats in the HTXD group(0.59±0.09/T-DAPK-1,P<0.05),M-TXD group(0.52±0.18/T-DAPK-1,P<0.05),L-TXD group(0.55±0.15/T-DAPK-1,P<0.05)TC-DAPK6 group(0.70±0.17/TDAPK-1,P<0.01)increased,indicating that the decreased DAPK-1 activity.There were no significant differences in the expression of P-DAPK-1(Ser308)/T-DAPK-1 in hippocampus of rats among H-TXD group,M-TXD group,L-TXD group,and TCDAPK6 group(P>0.05).6.Immunohistochemical detection of the effect of TXD and TC-DAPK6 on the expression of Bcl-2 in the hippocampus of diabetic rats:Compared with the Con group(22.0±4.6/FOV),the Bcl-2 level of the rats in the DM group(6.3±1.2/FOV)decreased(P<0.01).Compared with the DM group,the level of Bcl-2 in the hippocampus of HTXD group(24.3±3.1/FOV,P<0.01),M-TXD group(22.0±3.6/FOV,P<0.01),L-TXD group(18.0±5.0/FOV,P<0.05),TC-DAPK6 group(23.7±6.0/FOV,P<0.01)rats increased.There were no significant differences in the expression of Bcl-2 in the hippocampus of diabetic rats among H-TXD group,M-TXD group,L-TXD group,and TC-DAPK6 group(P>0.05).[Conclusion]1.DAPK-1 is involved in the occurrence and development of cognitive impairment in diabetic rats.2.TXD can reduce the activity of DAPK-1 in hippocampal neurons under high glucose environment,and increase the activity of neurons.TXD can protect hippocampal neurons and alleviate cognitive decline in diabetic rats by reducing DAPK-1 activity in the hippocampus of diabetic rats,down-regulating P-APP,phosphorylation Tau,and Caspase-3 levels,and up-regulating Bcl-2 levels.Compared with the medium and low dose TXD groups,the high dose TXD group had the best effect in down-regulating PAPP level.[Innovation]This study innovatively introduced DAPK-1 into DCI research,and observed that the activity and expression of DAPK-1 increased in hippocampal neurons under high glucose and diabetic rat hippocampus,and inhibiting the activity of DAPK-1 had significant effects on hippocampal neurons under high glucose and diabetic rats.This study found that TXD protects hippocampal neurons by reducing DAPK-1 activity,which is one of the protective mechanisms of TXD against cognitive impairment in diabetic rats,which lays the foundation for further digging into the scientific value of TXD in the future.