1.Physical observation and the Changes in body weights The observation on the state of rats in the morning and evening showed that there was no significant difference in the hair gloss, basic eating condition and defecation between the normal saline group and the other experimental groups, and the movement and activity of rats in the other groups were slightly poorer than those in the normal saline group, but there was no significant difference in them between normal saline group and the other groups (P > 0.05).
During the experiment, the body weight of all rats in each group increased in varying degrees, and there was no difference in it among the groups (Figure 1.), indicating that scopolamine hydrobromide and rLZ-8 should have no effect on the body weight of rats.
1. Morris water maze test
1.1 Training experiment
2.1.1 In the training experiment of Morris water maze test, the rats in the other groups except the normal saline group were given scopolamine hydrobromide 30min after the administration of rLZ-8 from the 8th day to 14th day of experiment, and the water maze test was performed 30min after the administration of scopolamine hydrobromide. the escape latency of reaching to the platform of rats in rLZ-8 groups, donepezil group and AD model group were significantly longer than those in the normal saline group (P < 0.05) on the 5th and 6th day. As shown in Table 1.
Table 1.
Escape latencies of rats in the training experiment of Morris water maze test (s, x±s)
Time (day)
|
Groups (n=10)
|
N.S
|
AD model
|
Donepezil
|
56 μg/kg rLZ-8
|
112 μg/kg rLZ-8
|
224 μg/kg rLZ-8
|
1
|
43.2±42.58
|
105.9±24.48*
|
110.3±20.11*
|
101.2±32.73*
|
96.8±42.04*
|
106.8±21.93*
|
2
|
24.3±31.97
|
102.3±31.97*
|
100.2±28.58*
|
103.0±33.56*
|
95.1±41.14*
|
91.6±40.89*
|
3
|
20.2±23.18
|
95.3±9.51*
|
92.4±28.24*
|
96.1±24.45*
|
83.1±15.08*
|
79.4±20.79*
|
4
|
10.8±9.51
|
92.1±19.51*
|
84.5±14.46*
|
88.8±15.69*
|
75.4±10.57*
|
87.0±18.38*
|
5
|
11.9±9.64
|
99.4±8.51*
|
46.0±12.46*#
|
56.2±7.08*##
|
67.8±12.09*#
|
80.9±8.03*#△△▲
|
6
|
8.9±6.60
|
92.1±9.51*
|
43.8±11.86*#
|
45.9±12.60*#
|
64.7±9.57*#△
|
81.4±5.27*#△△▲▲
|
* Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01.
#Compared with the model group, P<0.05; ##Compared with the model group, P<0.01. △Compared with donepezil group, P<0.05; △△ Compared with donepezil group, P<0.01. ▲Compared with 56μg/kg rLZ-8 group, P<0.05; ▲▲Compared with 56μg/kg rLZ-8 group, P<0.01.
|
2.1.2 In the training experiment of Morris water maze test, the rats in the other groups except the normal saline group were given scopolaminehydrobromide 30min after the administration of rLZ-8 from the 8th day to 14th day of experiment, and the water maze test was performed 30min after the administration of scopolamine hydrobromide. the swimming distance of rats in rLZ-8 groups, donepezil group and AD model group were significantly longer than those in the normal saline group (P< 0.05). As shown in Table 2.
Table 2.
Swimming distance results of rats in the training experiment of Morris water maze test (s, x±s)
Time
(day)
|
Groups (n=10)
|
Normal saline
|
AD model
|
Donepezil
|
56 μg/kg rLZ-8
|
112 μg/kg rLZ-8
|
224 μg/kg rLZ-8
|
1
|
1144.3±1045.5
|
3460.4±905.5*
|
3662.3±841.6*
|
3472.1±1173.2*
|
3026.4±1048.4*
|
3873.8±998.4*
|
2
|
646.0±726.6
|
3620.5±837.0*
|
3609.1±1173.6*
|
3726.6±1393.5*
|
3290.9±1445.3*
|
3584.8±1637.8*
|
3
|
520.4±562.2
|
3289.3±632.0*
|
3192.0±905.1*
|
3473.8±1195.2*
|
2974.4±1652.3*
|
3012.3±1965.2*
|
4
|
294.3±24.3
|
2939.2±435.7*
|
3082.9±725.2*
|
3231.4±684.3*
|
2709.2±885.2*
|
3028.6±918.6*
|
5
|
315.3±237.6
|
2907.3±235.8*
|
1519.4±413.9*#
|
2069.9±774.7*#
|
2235.9±783.8*#
|
3006.3±812.5*#△△▲
|
6
|
259.9±173.8
|
3010.5±470.1*
|
1541.2±106.7*#
|
1483.1±364.5*#
|
2174.7±630.7*#
|
2913.8±620.2*#△△▲▲
|
* Compared with the normal saline group, P<0.05; ** Compared with the normal saline group, P<0.01. # Compared with the model group, P<0.05; ## Compared with the model group, P<0.01. △ Compared with donepezil group, P<0.05; △△ Compared with donepezil group, P<0.01. ▲ Compared with 56μg/kg rLZ-8 group, P<0.05; ▲▲ Compared with 56μg/kg rLZ-8 group, P<0.01.
|
1.2 Space exploration test
The rats in the other groups except the normal saline group were given scopolamine hydrobromide 30min after the administration of rLZ-8 on the 14th day of experiment, and the space exploration test was conducted 30min after the administration of scopolamine hydrobromide. Compared with that in the normal saline group, the swimming time of the rats’ staying in the quadrant of the original platform was decreased in the model group, but there was no significant difference in it between the two groups (P>0.05), while the steering angle, the number of times passing through the location of the original platform and the number of times passing through the effective area of the original platform were significantly decreased (P<0.05). Compared with that in the model group, the steering angle, the number of times passing through the location of the original platform and the number of times passing through the effective area of the original platform were significantly increased in 112 μg/kg rLZ-8 group (P<0.05), the number of times passing through the location of the original platform and the number of times passing through the effective area of the original platform were significantly increased in 56ug/kg rLZ-8 group (P<0.05), and the steering angle and the number of times passing through the effective area of the original platform were significantly increased in donepezil group (P<0.05).as shown in Figure 1 and Table 3.
Table 3.
Space exploration test (s, x±s)
Groups (n=10)
|
Time in the second quadrant
|
Orientation angle
|
Number of passing through the location of original platform
|
Number of passing through the effective area
|
Normal saline
|
32.3±4.89
|
75.7±45.07
|
4.63±2.12#
|
6.63±1.93#
|
Model
|
24.62±5.81
|
56.4±42.64*
|
1.14±3.95*
|
4.57±3.50*
|
Donepezil
|
31.4±6.16
|
76.8±35.19#
|
1.25±2.22*
|
6.00±4.00#
|
56 μg/kg rLZ-8
|
38.3±10.59
|
67.3±32.94
|
2.38±1.93#
|
7.13±5.97#
|
112 μg/kg rLZ-8
|
28.4±7.71
|
85.3±32.82#
|
1.75±1.79*#
|
6.88±4.70#
|
224 μg/kg rLZ-8
|
30.4±2.67
|
61.3±31.08
|
1.38±1.58*
|
4.25±6.61*
|
* Compared with the normal saline group, P<0.05; # Compared with the model group, P<0.05.
lom the values of the Sco group: P<0.05. ## Results significantly differ from the values of the Sco group: P<0.01.
2. Biochemical Indexes
3.1 Biochemical indexes in the serum of rats
3.1.1 Acetylcholinesterase (TChE) activities
Compared with the normal saline group, the activity of TChE in the serum of rats in the model group was significantly higher (P < 0.01), compared with the model group,the 56 μg/kg rLZ-8 group was significantly lower (P < 0.05), and the donepezil group was significantly lower (P < 0.01). As shown in Table 4.
3.1.2 Monoamine oxidase (MAO) activities
Compared with the normal saline group,the activity of MAO in the serum of rats in the model group was significantly higher (P < 0.01), compared with the normal saline group, the 56 μg/kg, 112 μg/kg and 224 μg/kg rLZ-8, and donepezil groups was significantly higher (P < 0.01), compared with the model group, the 56 μg/kg, 112 μg/kg and 224 μg/kg rLZ-8, and donepezil groups was significantly lower (P < 0.01). As shown in Table 4.
Table 4.
TChE and MAO activities in the serum of rats (s, x±s)
Groups(n=10)
|
TChE (U/mgprot)
|
MAO (U/mgprot)
|
Normal saline
|
6.633±0.938
|
34.930±1.379
|
AD model
|
9.434±1.433**
|
55.737±1.534**
|
Donepezil
|
6.934±1.516##
|
48.771±1.806**##
|
56 μg/kg rLZ-8
|
6.035±1.426#
|
50.699±0.543**##△△
|
112 μg/kg rLZ-8
|
7.587±2.009
|
51.245±0.329**##△△▲
|
224 μg/kg rLZ-8
|
7.428±2.373
|
51.802±1.114**##△△▲
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01;# Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; △△Compared with donepezil group, P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05.
|
3.1.3 Superoxide dismutase (SOD) activities
Compared with the normal saline group, the activity of SOD in the serum of rats in the model group was significantly lower (P<0.01), and the 224 μg/kg rLZ-8 group was significantly lower (P<0.05); compared with the model group, the 56 μg/kg and 112 μg/kg rLZ-8 groups was significantly higher (P <0.01).As shown in Table 5.
3.1.4 Glutathione peroxidase (GSH-PX) activities
Compared with the normal saline group, the activity of GSH-PX in the serum of rats in the model group was significantly reduced (P < 0.01), and 56 μg/kg, 112 μg/kg and 224 μg/kg rLZ-8 groups was significantly reduced (P < 0.01); compared with the model group, the 56 μg/kg, 112 μg/kg and 224 μg/kg rLZ-8 groups was significantly increased (P < 0.01), and the donepezil group was also significantly increased (P < 0.01) . As shown in Table 5.
3.1.5 Malondialdehyde (MDA) contents
Compared with the normal saline group, the content of MDA in the serum of rats in the model group was significantly higher (P <0.01), and 112 μg/kg and 224 μg/kg rLZ-8 groups was significantly higher (P <0.05); compared with the model group, the donepezil and 56 μg/kg rLZ-8 groups (P <0.01), and 112 μg/kg rLZ-8 groups was significantly lower (P <0.05) . As shown in Table 5.
Table 5.
SOD and GSH-PX activities and MDA contents in the serum of rats (s, x±s)
Groups (n=10)
|
SOD (U/mgprot)
|
GSH-PX (U/mgprot)
|
MDA (nmol/ml)
|
Normal saline
|
217.08±46.61
|
1844.83±78.24
|
2.70±0.50
|
AD model
|
134.98±29.67**
|
1062.03±130.22**
|
5.35±1.66**
|
Donepezil
|
191.68±40.53##
|
1815.10±44.40##
|
3.67±1.21##
|
56 μg/kg rLZ-8
|
225.68±68.73##
|
1650.20±92.15**##△△
|
4.23±0.78##
|
112 μg/kg rLZ-8
|
218.26±78.09##
|
1538.18±98.54**##△△▲
|
3.82±1.22*#
|
224 μg/kg rLZ-8
|
165.44±50.46*▲
|
1405.71±107.40**##△△▲▲
|
3.39±2.03*
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.0; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; △△Compared with dopezil group, P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲ Compared with 56 μg/kg rLZ-8 group, P<0.01.
3.2. Biochemical indexes in the hippocampus and cerebral cortex of rats
3.2.1 Acetylcholinesterase (TChE) activities
Compared with the normal saline group, the activity of TChE in the hippocampus of rats in model group was significantly higher (P < 0.05), and the denepezil and 224 μg/kg rLZ-8 groups was significantly higher (P < 0.05); compared with the model group,
thedonepezil group was significantly lower (P < 0.05), the 56 μg/kg and 112 μg/kg rLZ-8 groups was significantly lower (P < 0.01). As shown in Table 6.
Compared with the normal saline group, the activity of AChE in the cerebral cortex of rats in the model group was significantly higher (P < 0.01); compared with the model group,the 56 μg/kg rLZ-8 group was significantly lower than (P < 0.05), and the donepezil group was significantly lower (P < 0.01). As shown in Table 7.
3.2.2 Choline acetyltransferase (ChAT) contents
Compared with the normal saline group, the content of ChAT in the hippocampus of rats was significantly lower in the model group (P < 0.01), compared with the model group,the donepezil and 56 μg/kg rLZ-8 groups was significantly higher (P < 0.01). As shown in Table 6.
Compared with the normal saline group, the content of ChAT in the cerebral cortex of rats was significantly lower in the model group (P < 0.05), compared with the model group, the The 224 ug/kg rLZ-8 and donepezil groups was significantly higher (P < 0.05), and the 56 μg/kg rLZ-8 group was significantly higher (P < 0.01). As shown in Table 7.
Table 6.
TChE activities and the ChAT contents in the hippocampus of rats (s, x±s)
Groups (n=10)
|
TChE activities (U/mgprot)
|
ChAT contents (IU/g)
|
Normal saline
|
0.205±0.055
|
261.71±33.37
|
AD model
|
0.280±0.137*
|
167.705±36.72**
|
Donepezil
|
0.195±0.097#
|
248.99±26.62##
|
56 μg/kg rLZ-8
|
0.197±0.058##
|
228.14±48.04##
|
112 μg/kg rLZ-8
|
0.185±0.048##
|
226.27±74.14
|
224 μg/kg rLZ-8
|
0.228±0.071*#
|
228.54±121.97
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01.
|
Table 7.
TChE activities and the ChAT contents in the cerebral cortex of rats (s, x ±s)
Groups (n=10)
|
TChE activities(U/mgprot)
|
ChAT contents(IU/g)
|
Normal saline
|
0.176±0.052
|
243.26±79.90
|
AD model
|
0.261±0.028**
|
165.80±58.91*
|
Donepezil
|
0.180±0.037##
|
194.24±61.06#
|
56 μg/kg rLZ-8
|
0.197±0.138#
|
172.98±23.95##
|
112 μg/kg rLZ-8
|
0.222±0.057
|
174.78±57.85
|
224 μg/kg rLZ-8
|
0.242±0.168
|
202.59±63.97#
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01.
|
3.2.3 Superoxide dismutase (SOD) activities
Compared with the normal saline group, the activity of SOD in the hippocampus of rats decreased significantly in the model group (P < 0.05), compared with the model group, the donepezil, 56 μg/kg, 224 μg/kg rLZ-8 groups was significantly increased (P < 0.01). As shown in Table 8.
Compared with the normal saline group, the activity of SOD in the cerebral cortex of rats was significantly lower in the model group (P < 0.01) and significantly higher in donepezil group (P < 0.01), compared with the model group, the donepezil, 56 μg/kg and 112 μg/kg rLZ-8 groups was significantly higher (P < 0.05) . As shown in Table 9.
3.2.4 Glutathione peroxidase (GSH-PX) activities
Compared with the normal saline group, the activity of GSH-PX in the hippocampus of rats was significantly decreased in the model group (P < 0.01) and significantly decreased in 224 μg/kg rLZ-8 group (P < 0.01), compared with the model group, the donepezil and 56 μg/kg rLZ-8 groups was significantly increased (P < 0.01), and 112 μg/kg rLZ-8 group was significantly increased (P < 0.05) . As shown in Table 8.
Compared with the normal saline group, the activity of GSH-PX in the cerebral cortex of rats was significantly lower in the model group (P < 0.05), compared with the model group, the 112 μg/kg rLZ-8 group was significantly higher (P < 0.05), and the donepezil group was significantly higher (P < 0.01) . As shown in Table 9.
3.2.5 Malondialdehyde (MDA) contents
Compared with the normal saline group, the content of MDA in the hippocampus of rats was significantly increased in the model group (P < 0.01), compared with the model group, the donoperazide and 56 μg/kg rLZ-8 groups was significantly lower (P < 0.05) ,and the 112 μg/kg rLZ-8 group was significantly lower (P < 0.01). As shown in Table 8.
Compared with the normal saline group, the content of MDA in the cerebral cortex of rats was significantly increased in the model group (P < 0.01); compared with the model group, the donepezil and 56 μg/kg rLZ-8 groups was significantly decreased (P < 0.05), and 112 and 224 μg/kg rLZ-8 groups was significantly decreased (P < 0.01) . As shown in Table 9.
Table 8.
SOD and GSH-PX activities and MDA contents in the hippocampus of rats (s, x±s)
Groups (n=10)
|
SOD (U/mgprot)
|
GSH-PX (U/mgprot)
|
MDA (nmol/ml)
|
Normal saline
|
85.25±35.55
|
38.99±6.45
|
0.34±0.02
|
AD model
|
51.90±21.59*
|
27.64±5.39**
|
0.41±0.02**
|
Donepezil
|
79.02±37.40##
|
35.99±3.53##
|
0.34±0.04#
|
56 μg/kg rLZ-8
|
63.09±12.72##
|
34.49±1.87##
|
0.36±0.02##
|
112 μg/kg rLZ-8
|
58.82±18.11
|
33.71±5.00#
|
0.36±0.03##
|
224 μg/kg rLZ-8
|
58.85±11.10##△
|
30.65±5.30**
|
0.35±0.08
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; .
Table 9.
SOD and GSH-PX activities and MDA contents in the cerebral cortex of rats (s, x±s)
Groups (n=10)
|
SOD (U/mgprot)
|
GSH-PX (U/mgprot)
|
MDA (nmol/ml)
|
Noemal saline
|
129.58±14.22
|
40.65±9.65
|
0.90±0.13
|
AD model
|
115.21±17.68**
|
33.64±17.67*
|
1.11±0.09**
|
Donepezil
|
135.11±31.22**##
|
40.81±10.10#
|
0.91±0.28#
|
56 μg/kg rLZ-8
|
137.91±21.74#
|
40.64±5.12
|
0.92±0.41#
|
112 μg/kg rLZ-8
|
135.23±25.26#
|
36.97±6.16##
|
0.94±0.30##
|
224 μg/kg rLZ-8
|
110.22±10.21#△▲▲
|
36.54±3.01▲
|
0.99±0.15##
|
*Compared with the normal saline, P<0.05; **Compared with the normal saline, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; △△Compared with donepezil group, P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲Compared with 56 μg/kg rLZ-8 group, P<0.01.
3.2.6 Catalase (CAT) activities
Compared with the normal saline group,the activity of CAT in the hippocampus of rats was significantly lower in the model, donepezil and 112 μg/kg rLZ-8 groups (P < 0.01), compared with the model group, the donepezil and 56 μg/kg rLZ-8 a groups was significantly higher (P < 0.01), and the 224 μg/kg group was significantly higher (P < 0.05). As shown in Table 10.
Compared with the normal saline group,the activity of CAT in the cerebral cortex of rats in the model group was significantly lower (P < 0.05), compared with the model group,the donepezil group was significantly higher (P < 0.01), and the 56 μg/kg and 112 μg/kg rLZ-8 groups was significantly higher (P < 0.05). As shown in Table 11.
3.2.7 Nitric oxide synthase (NOS) contents
The contents of TNOS and iNOS in the hippocampus of rats in the model group were significantly higher than those in the normal saline group (P <0.05), the content of iNOS in the hippocampus of rats in donepezil, 56 μg/kg rLZ-8 and 112 μg/kg rLZ-8 groups was significantly lower than that in the model group (P <0.05), and the content of iNOS in 56 μg/kg rLZ-8 group was significantly lower than that in the model group (P <0.05). As shown in Table10 .
The contents of TNOS and iNOS in the cerebral cortex of rats in the model group were significantly higher than those in the normal saline group (P <0.05), and the contents of TNOS and iNOS in donepezil and 56 μg/kg rLZ-8 groups were significantly lower (P <0.05). As shown in Table 11.
Table 10.
TNOS and iNOS contents and CAT activities in the hippocampus of rats (s, x±s)
Groups (n=10)
|
CAT (U/mgprot)
|
TNOS (U/mgprot)
|
iNOS (U/mgprot)
|
Normal saline
|
0.637±0.189
|
0.982±0.259
|
0.577±0.166
|
AD model
|
0.264±0.127**
|
1.237±0.185*
|
0.783±0.175*
|
Donepezil
|
0.411±0.137*##
|
0.987±0.299#
|
0.560±0.153##
|
56 μg/kg rLZ-8
|
0.562±0.206##
|
0.935±0.213##
|
0.548±0.199#
|
112 μg/kg rLZ-8
|
0.374±0.170**▲
|
0.995±0.283#
|
0.608±0.256
|
224 μg/kg rLZ-8
|
0.627±0.449#
|
1.268±1.117▲
|
0.715±0.328
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; ▲ Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲.
Table 11.
TNOS and iNOS contents and CAT activities in the cerebral cortex of rats (s, x±s)
Groups (n=10)
|
CAT (U/gprot)
|
TNOS (U/mgprot)
|
iNOS (U/mgprot)
|
Normal saline
|
0.233±0.125
|
1.557±0.329
|
0.549±0.198
|
AD model
|
0.129±0.038*
|
1.953±0.446*
|
0.794±0.284*
|
Donepezil
|
0.273±0.112##
|
1.499±0.368#
|
0.548±0.174#
|
56μg/kg rLZ-8
|
0.216±0.082#
|
1.446±0.389#
|
0.512±0.157#
|
112 μg/kg rLZ-8
|
0.194±0.073#
|
1.715±0.552
|
0.711±0.425
|
224 μg/kg rLZ-8
|
0.178±0.137
|
1.757±0.521
|
0.732±0.386
|
*Compared with the normal saline group, P<0.05;#Compared with the model group, P<0.05; ##Compared with the model group, P<0.01.
3. Cytokines Detected by ELISA
4.1 Cytokines in the serum of rats
4.1.1 Tumor Necrosis Factor α (TNF-α)
Compared with the normal saline group, the content of TNF-α in the serum of rats did not change significantly in the model group, but the content of TNF-α increased slightly in the donepezil and rLZ-8-treated groups, showing no difference; compared with 56 μg/kg rLZ-8 group, the 224 μg/kg rLZ-8 group was significantly lower (P < 0.05). As shown in Table 12.
4.1.2 Interleukin 6 (IL-6) contents
Compared with the normal saline group, the content of IL-6 in the serum of rats was significantly higher in 56 μg/kg rLZ-8 group (P < 0.05); compared with 56 μg/kg rLZ-8 group, the 112 μg/kg rLZ-8 group was significantly lower (P < 0.05), and the in 224 μg/kg rLZ-8 group was significantly lower (P < 0.01). As shown in Table 12.
4.1.3 Interlukin 1β (IL-1β) contents
Compared with that normal saline group, there was no significant change in the content of IL-1β in the serum of rats in the other experimental groups; compared with the model group, the 112 and 224 μg/kg rLZ-8 groups was significantly lower (P < 0.05); compared with donepezil group, the 56 μg/kg rLZ-8 group was significantly higher (P < 0.05), and the 112 and 224 μg/kg rLZ-8 groups was significantly lower (P < 0.01). As shown in Table 12.
Table 12.
TNF-α, IL-6 and IL-1β contents in the serum of rats (s, x±s)
Groups(n=10)
|
TNF-α(ng/L)
|
IL-6 (pg/ml)
|
IL-1β (pg/ml)
|
Normal saline
|
294.588±36.689
|
113.766±19.176
|
48.003±18.864
|
AD model
|
294.516±50.525
|
127.669±21.464
|
52.164±8.917
|
Donepezil
|
315.411±124.049
|
99.966±20.388#
|
44.949±11.220
|
56 μg/kg rLZ-8
|
336.733±51.569
|
143.790±32.699*△
|
56.551±9.510△
|
112 μg/kg rLZ-8
|
310.063±45.082
|
113.729±19.594▲
|
43.736±6.730#▲▲
|
224 μg/kg rLZ-8
|
283.463±39.842▲
|
85.545±22.363*##▲▲§
|
39.859±11.560#▲▲
|
*Compared with the normal group, P<0.05; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05;▲Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲Compared with 56 μg/kg rLZ-8 group, P<0.01; §Compared with 112 μg/kg rLZ-8 group, P<0.05
4.2 Cytokines in the hippocampus and cerebral cortex of rats
4.2.1 Tumor necrosis factor α (TNF-α) contents
Compared with the normal saline group, the content of TNF-α in the hippocampus of rats was not significantly different in the other experimental groups, compared with the model group, the 224 μg/kg rLZ-8 group was significantly lower (P < 0.05). As shown in Table 13.
Compared with the normal saline group, the content of TNF-α in the cerebral cortex of rats was significantly lower in donepezil group (P < 0.01), while the other groups was not significantly different; compared with the model group, the donepezil group was significantly reduced (P < 0.01); compared with donepezil group, the three groups treated with rLZ-8 was significantly elevated (P < 0.01); compared with 56 μg/kg rLZ-8 group, the 112 μg/kg rLZ-8 group was significantly elevated (P < 0.05); compared with 112 μg/kg rLZ-8 group, the 224 μg/kg rLZ-8 group was significantly reduced (P < 0.05). As shown in Table 14.
4.2.2 Interleukin 6 (IL-6) contents
Compared with the normal saline group, the content of IL-6 in the hippocampus of rats was significantly reduced indonepezil and 224 μg/kg rLZ-8 group (P < 0.01), and the 56 μg/kg rLZ-8 group was also significantly reduced (P < 0.05); compared with the model group, the donepezi and 224 μg/kg rLZ-8 groups was significantly reduced (P < 0.01), and 56 μg/kg rLZ-8 group decreased significantly (P < 0.05); compared with donepezil, 56 μg/kg rLZ-8 and 112 μg/kg rLZ-8 groups, the 224 μg/kg rLZ-8 group was significantly decreased (P < 0.01). As shown in Table 13.
Compared with the normal saline group, the content of IL-6 in the cerebral cortex of rats decreased significantly in 56 and 224 μg/kg rLZ-8 groups (P < 0.05), the other groups did not change significantly (P > 0.05); compared with the model group, the in56 and 224 μg/kg rLZ-8 groups was significantly decreased (P < 0.01); compared with 56 μg/kg rLZ-8 group, the 112 μg/kg rLZ-8 group was significantly increased (P < 0.01); compared with 112 μg/kg rLZ-8 group, the 224 μg/kg rLZ-8 group was significantly decreased (P < 0.01). As shown in Table 14.
4.2.3 Interlukin 1β (IL-1β) contents
As shown in Table 13, there was no significant difference in the content of IL-1β in the hippocampus of rats among the different experimental groups;
Compared with the normal saline group, the content of IL-1β in the cerebral cortex of rats was significantly lower in donepezil group (P < 0.01); compared with the model group, the donepezil and 56 μg/kg rLZ-8 groups was significantly lower (P < 0.01), and in 224 μg/kg rLZ-8 group (P < 0.05); compared with donepezil group, the three rLZ-8-treated groups was significantly lower (P < 0.01). As shown in Table 14
Table 13.
TNF-α, IL-6 and IL-1β contents in the hippocampus of rats (s, x±s)
Groups (n=6)
|
TNF-α (ng/mg)
|
IL-6 (ng/mg)
|
IL-1β (ng/mg)
|
Normal saline
|
3.720±0.392
|
5.027±0.618
|
0.876±0.192
|
AD model
|
3.996±0.384
|
5.511±0.784
|
1.024±0.267
|
Donepezil
|
3.481±0.473
|
3.768±0.428**##
|
0.762±0.153
|
56 μg/kg rLZ-8
|
3.482±0.454
|
4.258±0.438*#
|
0.813±0.053
|
112 μg/kg rLZ-8
|
3.968±0.818
|
4.279±1.057
|
0.900±0.199
|
224 μg/kg rLZ-8
|
3.347±0.285#
|
3.102±0.503**##△△▲§§
|
0.848±0.044
|
*Compared with the normal saline group, P<0.05; **Compared with the normal saline group, P<0.01; # Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; △△Compared with donepezil group, P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05; §§Compared with 112 μg/kg rLZ-8 group, P<0.05.
Table 14.
TNF-α, IL-6 and IL-1β in the cerebral cortex of rats (s, x±s)
Groups (n=10)
|
TNF-α (ng/mg)
|
IL-6 (ng/mg)
|
IL-1β (ng/mg)
|
Normal saline
|
3.431±0.476
|
4.206±0.854
|
0.768±0.157
|
AD model
|
3.407±0.262
|
5.226±0.680
|
0.924±0.142
|
Donepezil
|
2.106±0.159**##
|
3.506±0.552#
|
0.477±0.101**##
|
56 μg/kg rLZ-8
|
3.106±0.341△△
|
3.052±0.456*##
|
0.691±0.057##△△
|
112 μg/kg rLZ-8
|
4.118±0.849△△▲
|
4.732±0.632△△▲▲
|
0.871±0.186△△
|
224 μg/kg rLZ-8
|
3.196±0.346△△§
|
3.021±0.090*##§§
|
0.725±0.081#△△
|
**Compared with the normal saline group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01;△△Compared with donepezil group, P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲Compared with 56 μg/kg rLZ-8 group, P<0.01; §Compared with 112 μg/kg rLZ-8 group, P<0.05; §§Compared with 112 μg/kg rLZ-8 group, P<0.05.
4.2.4 Glial-derived neurotrophic factor (GDNF) contents
Compared with the normal saline group, the content of GDNF in the hippocampus of rats the model group was significantly lower than the normal saline group (P < 0.05), compared with the model group,the 112 and 224μg/kg rLZ-8 groups was significantly higher (P < 0.05), compared with the 56 μg/kg rLZ-8 group, the 112 μg/kg rLZ-8 group was significantly higher (P < 0.05). As shown in Table 15.
Compared with the normal saline group, the content of GDNF in the cerebral cortex of rats was not significantly different in the other experimental groups, compared with in the model group,the 56 and 224 μg/kg rLZ-8 groups was significantly lower (P < 0.05), compared with the 56 μg/kg rLZ-8 group,the 112 μg/kg rLZ-8 groups was significantly higher (P < 0.05), compared with the 112 μg/kg rLZ-8 group,the 224 μg/kg rLZ-8 groups was significantly lower (P < 0.01), As shown in Table 16.
4.2.5 Brain-derived neurotrophic factor (BDNF) contents
Compared with the normal saline group, the content of BDNF in the hippocampus of rats in 224 μg/kg rLZ-8 group was significantly higher (P < 0.05)), compared with the model group,the donepezil group was significantly higher (P < 0.05) and the 224 μg/kg rLZ-8 group was significantly higher (P < 0.01), compared with the 56 μg/kg rLZ-8 group, the 224 μg/kg rLZ-8 group was significantly higher (P < 0.01), compared with the 112 μg/kg rLZ-8 group, the 224 μg/kg rLZ-8 group was significantly higher (P < 0.05),As shown in Table 15.
Compared with the normal saline group, the content of BDNF in the cerebral cortex of rats in the model group was significantly lower (P < 0.01) and the 112 μg/kg rLZ-8 group was significantly higher (P < 0.01); Compared with the model group, the donepezil and 112 μg/kg rLZ-8 groups was significantly increased (P < 0.01), and the224 μg/kg rLZ-8 group was also significantly increased (P < 0.05); compared with 112 μg/kg rLZ-8 group, the donepezil, 56 μg/kg rLZ-8 and 224 μg/kg rLZ-8 groups was significantly decreased (P < 0.01). As shown in Table 16
Table 15.
GDNF and BDNF contents in the hippocampus of rats (s, x±s)
Groups (n=10)
|
GDNF (ng/mg)
|
BDNF (ng/mg)
|
Normal saline
|
2.537±0.370
|
2.942±0.511
|
AD model
|
2.145±0.059*
|
2.643±0.326
|
Donepezil
|
2.313±0.282
|
3.384±0.499#
|
56 μg/kg rLZ-8
|
2.176±0.117
|
2.511±0.232△
|
112 μg/kg rLZ-8
|
2.561±0.304#▲
|
3.036±0.565
|
224 μg/kg rLZ-8
|
2.361±0.171#
|
3.776±0.352*##▲▲§
|
*Compared with the normal saline group, P<0.05; P<0.01; # Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲Compared with 56 μg/kg rLZ-8 group, P<0.01; §Compared with 112 μg/kg rLZ-8group, P<0.05;.
Table 16.
GDNF and BDNF contents in the cerebral cortex of rats (s, x±s)
Groups (n=10)
|
GDNF(ng/mg)
|
BDNF(ng/mg)
|
Normal saline
|
1.725±0.462
|
2.918±0.131
|
AD model
|
1.990±0.109
|
2.476±0.155**
|
Donepezil
|
1.796±0.099
|
3.223±0.298##
|
56 μg/kg rLZ-8
|
1.801±0.067#
|
2.898±0.454
|
112 μg/kg rLZ-8
|
2.312±0.269△▲
|
4.166±0.204**##△△▲▲
|
224 μg/kg rLZ-8
|
1.675±0.127#§§
|
2.885±0.159#§§
|
**Compared with the normal saline group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01; △Compared with donepezil group, P<0.05; △△Compared with donepezil group, P<0.01; ▲Compared with 56 μg/kg rLZ-8 group, P<0.05; ▲▲Compared with 56 μg/kg rLZ-8 group, P<0.01; §§ Compared with 112 μg/kg rLZ-8 group, P<0.01.
4.Organ Indexes
Compared with the normal saline group, the thymus index of rats in the model group decreased significantly (P < 0.05), and there was no significant difference in the other organ indexes between the normal saline group and model group (P>0.05), indicating that scopolamine hydrobromide had no significant effect on the index of organs except the thymus. Compared with the normal saline group, the lung index and the spleen index of rats increased significantly in 56 μg/kg rLZ-8 group (P < 0.05), the spleen index decreased significantly in 112 μg/kg rLZ-8 group (P < 0.05), and the spleen index increased significantly in 224 μg/kg rLZ-8 group (P < 0.05). Compared with that in the model group, the lung index, the spleen index and the thymus index increased significantly in 56 μg/kg rLZ-8 group (P < 0.05), the spleen index decreased significantly in 112 μg/kg rLZ-8 group (P < 0.05), and the spleen index and the thymus index increased significantly in 224 μg/kg rLZ-8 group (P < 0.05). After the administration of scopolamine hydrobromide for one week, its effects on the organ indexes of rats were not significant, but the administration of different dosages of scopolamine hydrobromide for two weeks showed some effects on the spleen and thymus of rats because an immunomodulatory protein could regulate the immune system to a certain extent, suggesting that the changes in the immune organ indexes of rats during the experiment should be a manifestation of the regulation of the rats’ autoimmunity system. As shown in Table 17.
Table 17.
Organ indexes (s, x±s)
Groups (n=10)
|
|
Normal saline
|
Model
|
Donepezil
|
56 μg/kg rLZ-8
|
112 μg/kg rLZ-8
|
224 μg/kg rLZ-8
|
Heart index
|
0.33±0.007
|
0.36±0.004
|
0.35±0.004
|
0.34±0.004
|
0.33±0.005
|
0.34±0.004
|
Liver index
|
3.75±0.067
|
3.51±0.045
|
3.58±0.060
|
3.60±0.050
|
3.64±0.055
|
3.54±0.055
|
Lung index
|
0.44±0.004
|
0.43±0.004
|
0.47±0.006
|
0.49±0.009*#
|
0.45±0.004
|
0.50±0.019
|
Spleen index
|
0.23±0.001
|
0.23±0.001
|
0.26±0.001
|
0.28±0.001*#
|
0.20±0.001*#
|
0.41±0.002*#
|
Thymus index
|
0.19±0.001
|
0.16±0.001*
|
0.19±0.001
|
0.21±0.001#
|
0.19±0.001
|
0.24±0.001#
|
Kidney index
|
0.71±0.010
|
0.71±0.007
|
0.70±0.010
|
0.68±0.017
|
0.70±0.006
|
0.68±0.012
|
* Compared with the normal saline group, P<0.05; # Compared with the model group, P<0.05.