Protective Effect of Recombinant Ganoderma Lucidum Immunomodulatory Protein (rLZ-8) Against Scopolamine-Induced Alzheimer's Disease in Rats

Backgroud: Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in the elderly, seriously threatening the health of the elderly. In this study, the protective effect of recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) against the scopolamine-induced Alzheimer's disease (AD) in rats was studied for the rst time. Methods: Male Wistar rats in rLZ-8-treated groups were intraperitoneally injected with 56 μg/kg, 112 μg/kg, and 224 μg/kg rLZ-8, respectively, those in donepezil group were intraperitoneally injected with 1.0mg/kg donepezil, and those in the normal saline group were intraperitoneally injected with an equal volume of normal saline, successively for 14 days. On the 7 th day after the administration, the learning and memory ability of rats was observed by Morris water maze test, and the biochemical indexes and cytokines in the serum and brain tissues of the rats were detected after the behaviour test for investigating the protective mechanism of rLZ-8 against the scopolamine-induced AD in rats. Results: In the water maze test, compared with that in the model group, the escape latency and the swimming distance of rats on the 5 th and 6 th day were signicantly shortened in the three rLZ-8-treated groups. In the space exploration test, compared with that in the model group, the number of passing through the location of original platform was signicantly increased and the time of rats’ staying in the second quadrant was signicantly prolonged in the three rLZ-8-treated groups, and furthermore, the detectionresults related to Alzheimer's disease in the serum, hippocampus and cerebral cortex of ratsindicated that rLZ-8 could improve the contents or activities of the related indexes. Conclusion: In rlZ-8 could signicantly improve the learning and memory ability of AD rats, and the possible mechanism was to improve the learning and memory ability by protecting the cholinergic system. could signicantly improve the learning and memory impairment induced by scopolamine hydrobromide. Free radical damage is the pathological basis of many diseases, and induced mainly by the uncontrollable and abnormal free radical reaction in cells, resulting in serious damages to the structure and function of cells, and free radicals are toxic products that are produced by organisms and can damage themselves, causing lipid peroxidation to produce lipid peroxides. The results in this study showed that the oxygen free radicals were scavenged to some extent in rats in the different rLZ-8-treated groups. NO (nitric oxide) as a vasoactive factor and a special neurotransmitter in the brain is involved in regulating the physiological process of cerebral blood ow, and learning and memory. The contents of total TNOS andiNOS were detected in this experiment, and the results showed that the different doses of rLZ-8 could regulate the activity of NOS enzyme and improve the learning and memory of rats with Alzheimer’s disease.

ability is the degeneration of cholinergic neurons in BF. The molecular formula of L-scopolamine is C 17 H 21 NO 4 ,that it is a scopolamine-type alkaloid and one of the strongest pharmacological agents in belladonna, and it can be used to block parasympathetic nerve and also used as a central nervous system inhibitor. Scopolamine blocks the binding of postsynaptic acetylcholine to its receptors by competitively binding to M receptors in the central nervous system to reduce the role of acetylcholine associated with learning and memory, inducing the AD animal model with learning and memory impairment [16,17] , and the model as one of the commonly used AD models has been widely used in the experiment of drugs used for the treatment of AD.
Based on the previous studies, the effect of recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) on the learning and memory ability of rats with AD, and its underlying mechanisms were investigated in this study, which may provide a basis for the development of drugs for the treatment of AD.

Animal Grouping
After they were acclimatized to the laboratory environment for a week, 60 rats were randomly divided into 6 groups, namely normal saline group, AD model group, donepezil group, 56 μg/kg rLZ-8 group, 112 μg/kg rLZ-8 group, and 224 μg/kg rLZ-8 group, 10 rats in each group.

Drug Administration and Animal Model Preparation
Rats in the normal saline group and model group were intraperitoneally injected with saline twice a day, at 8:00 and 16:00, respectively, successively for 14 days (day1-day14), those in donepezil group (P) were administrated intragastrically with 1.0 mg/kg donepezil at 8:00 once a day, successively for 14 days (day1-day14), and those in the three rLZ-8 groups were intraperitoneally injected with the corresponding doses of rLZ-8 twice a day at 8:00 and 16:00, respectively, successively for 14 days (day1-day14). Except those in the saline group, all rats in the other groups were intraperitoneally injected with 1.5mg/kg scopolamine hydrobromide at 10:00 once a day, successively from day 8 to day14 (day8-day14).

Observation Indexes
3.1 Physical observation: The body weight of rats was weighed with a balance and recorded every day. The state of the rats was observed twice a day in the morning and afternoon, respectively, including the hair color, eating, defecation, movement, and sensitivity of rats.
3.2 Morris water maze test: Morris water maze test was used to evaluate the learning and memory of rats, lasting 7ds. Training test: The training test lasted 6 days and all rats were trained four times a day, in which the rats were placed into the water at the different entry water points each time for observing the escape latency, that is, the time required for the rats to climb the platform from the time when they were placed into the water within 120s, and the rats were allowed to stay on the platform for 10s; if the rats did not nd the platform within 120s, the experimenter guided them to stay on the platform for 10s, and the escape latency was recorded as 120s; the swimming distance of rats' from the entry water points to nding the platform was recorded as swimming distance. Space exploration test: On the 7 th day of the test, the platform was removed for observing the number of times that the rats swam over the original platform, the time of swimming in the original place where the platform was placed, and the steering angle of rats. Each space exploration test lasted 120 s, in which the latency for the rats to nd the original place, the swimming distance for the rats to swim to the original place and the times of rats' swimming across the original place were recorded as their memory results.
3.3 Detection of biochemical indexes: The rats were anesthetized with chloral hydrate immediately after the Morris water maze test. After the blood samples were taken from their abdominal aorta, their whole brain, hippocampus and cerebral cortex samples were taken immediately, and then the heart, liver, spleen, lung, kidney and thymus were taken rapidly. The blood sample was centrifuged at 3000 rpm for 10 minutes to obtain the supernatants, and the serum acetylcholinesterase (AchE), monoamine oxidase (MAO), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities, and malondialdehyde (MDA) contents were detected according to the instructions of assay kits. After weighing, the hippocampus and cerebral cortex were added with normal saline at the volume ratio of 1:9 and homogenized to be prepared into the homogenates, and then the homogenates were centrifuged at 2,500 rpm for 15 minutes to obtain the supernatants. the AchE, SOD, GSH-PX and choline acetyltransferase (ChAT), catalase (CAT), ATP enzyme, nitric oxide synthase activities, and MDA, tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), interleukin-β (IL-1β), glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) contents in the supernatants were detected by following the instructions of the kits. The body weights and organ weights of rats were also weighed, respectively, for the calculation of their organ indexes.

Statistical Analysis
The data were statistically analyzed using SPSS16.0 and expressed as means±SD (x±s). The difference between two groups was compared by t-test and P < 0.05 was considered signi cant.

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 signi cant 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 signi cant 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 8 th day to 14 th 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 signi cantly longer than those in the normal saline group (P < 0.05) on the 5th and 6th day. As shown in Table 1. 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 signi cantly longer than those in the normal saline group (P< 0.05). As shown in Table 2. 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 signi cant 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 signi cantly 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 signi cantly 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 signi cantly 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 signi cantly increased in donepezil group (P<0.05).as shown in Figure 1 and Table 3. Compared with the normal saline group, the activity of TChE in the serum of rats in the model group was signi cantly higher (P < 0.01), compared with the model group,the 56 μg/kg rLZ-8 group was signi cantly lower (P < 0.05), and the donepezil group was signi cantly lower (P < 0.01). As shown in Table 4.

Monoamine oxidase (MAO) activities
Compared with the normal saline group,the activity of MAO in the serum of rats in the model group was signi cantly 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 signi cantly 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 signi cantly lower (P < 0.01). As shown in Table 4.

Superoxide dismutase (SOD) activities
Compared with the normal saline group, the activity of SOD in the serum of rats in the model group was signi cantly lower (P<0.01), and the 224 μg/kg rLZ-8 group was signi cantly lower (P<0.05); compared with the model group, the 56 μg/kg and 112 μg/kg rLZ-8 groups was signi cantly higher (P <0.01).As shown in Table 5.

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 signi cantly reduced (P < 0.01), and 56 μg/kg, 112 μg/kg and 224 μg/kg rLZ-8 groups was signi cantly reduced (P < 0.01); compared with the model group, the 56 μg/kg, 112 μg/kg and 224 μg/kg rLZ-8 groups was signi cantly increased (P < 0.01), and the donepezil group was also signi cantly increased (P < 0.01) . As shown in Table 5.

Malondialdehyde (MDA) contents
Compared with the normal saline group, the content of MDA in the serum of rats in the model group was signi cantly higher (P <0.01), and 112 μg/kg and 224 μg/kg rLZ-8 groups was signi cantly 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 signi cantly lower (P <0.05) . As shown in Table 5. Compared with the normal saline group, the activity of TChE in the hippocampus of rats in model group was signi cantly higher (P < 0.05), and the denepezil and 224 μg/kg rLZ-8 groups was signi cantly higher (P < 0.05); compared with the model group, thedonepezil group was signi cantly lower (P < 0.05), the 56 μg/kg and 112 μg/kg rLZ-8 groups was signi cantly 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 signi cantly higher (P < 0.01); compared with the model group,the 56 μg/kg rLZ-8 group was signi cantly lower than (P < 0.05), and the donepezil group was signi cantly lower (P < 0.01). As shown in Table 7.

Choline acetyltransferase (ChAT) contents
Compared with the normal saline group, the content of ChAT in the hippocampus of rats was signi cantly lower in the model group (P < 0.01), compared with the model group,the donepezil and 56 μg/kg rLZ-8 groups was signi cantly 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 signi cantly lower in the model group (P < 0.05), compared with the model group, the The 224 ug/kg rLZ-8 and donepezil groups was signi cantly higher (P < 0.05), and the 56 μg/kg rLZ-8 group was signi cantly higher (P < 0.01). As shown in Table 7.

Superoxide dismutase (SOD) activities
Compared with the normal saline group, the activity of SOD in the hippocampus of rats decreased signi cantly in the model group (P < 0.05), compared with the model group, the donepezil, 56 μg/kg, 224 μg/kg rLZ-8 groups was signi cantly 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 signi cantly lower in the model group (P < 0.01) and signi cantly 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 signi cantly higher (P < 0.05) . As shown in Table 9.

Glutathione peroxidase (GSH-PX) activities
Compared with the normal saline group, the activity of GSH-PX in the hippocampus of rats was signi cantly decreased in the model group (P < 0.01) and signi cantly 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 signi cantly increased (P < 0.01), and 112 μg/kg rLZ-8 group was signi cantly 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 signi cantly lower in the model group (P < 0.05), compared with the model group, the 112 μg/kg rLZ-8 group was signi cantly higher (P < 0.05), and the donepezil group was signi cantly higher (P < 0.01) . As shown in Table 9.

Malondialdehyde (MDA) contents
Compared with the normal saline group, the content of MDA in the hippocampus of rats was signi cantly increased in the model group (P < 0.01), compared with the model group, the donoperazide and 56 μg/kg rLZ-8 groups was signi cantly lower (P < 0.05) ,and the 112 μg/kg rLZ-8 group was signi cantly 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 signi cantly increased in the model group (P < 0.01); compared with the model group, the donepezil and 56 μg/kg rLZ-8 groups was signi cantly decreased (P < 0.05), and 112 and 224 μg/kg rLZ-8 groups was signi cantly decreased (P < 0.01) . As shown in Table 9.  Compared with the normal saline group,the activity of CAT in the hippocampus of rats was signi cantly 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 signi cantly higher (P < 0.01), and the 224 μg/kg group was signi cantly 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 signi cantly lower (P < 0.05), compared with the model group,the donepezil group was signi cantly higher (P < 0.01), and the 56 μg/kg and 112 μg/kg rLZ-8 groups was signi cantly higher (P < 0.05). As shown in Table 11.

Nitric oxide synthase (NOS) contents
The contents of TNOS and iNOS in the hippocampus of rats in the model group were signi cantly 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 signi cantly lower than that in the model group (P <0.05), and the content of iNOS in 56 μg/kg rLZ-8 group was signi cantly 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 signi cantly 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 signi cantly lower (P <0.05). As shown in Table 11. Compared with the normal saline group, the content of TNF-α in the serum of rats did not change signi cantly 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 signi cantly lower (P < 0.05). As shown in Table 12.

Interlukin 1β (IL-1β) contents
Compared with that normal saline group, there was no signi cant 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 signi cantly lower (P < 0.05); compared with donepezil group, the 56 μg/kg rLZ-8 group was signi cantly higher (P < 0.05), and the 112 and 224 μg/kg rLZ-8 groups was signi cantly lower (P < 0.01). As shown in Table 12. Compared with the normal saline group, the content of TNF-α in the hippocampus of rats was not signi cantly different in the other experimental groups, compared with the model group, the 224 μg/kg rLZ-8 group was signi cantly 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 signi cantly lower in donepezil group (P < 0.01), while the other groups was not signi cantly different; compared with the model group, the donepezil group was signi cantly reduced (P < 0.01); compared with donepezil group, the three groups treated with rLZ-8 was signi cantly elevated (P < 0.01); compared with 56 μg/kg rLZ-8 group, the 112 μg/kg rLZ-8 group was signi cantly elevated (P < 0.05); compared with 112 μg/kg rLZ-8 group, the 224 μg/kg rLZ-8 group was signi cantly reduced (P < 0.05). As shown in Table 14.

Interlukin 1β (IL-1β) contents
As shown in Table 13, there was no signi cant 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 signi cantly lower in donepezil group (P < 0.01); compared with the model group, the donepezil and 56 μg/kg rLZ-8 groups was signi cantly 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 signi cantly 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)

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 signi cantly lower than the normal saline group (P < 0.05), compared with the model group,the 112 and 224μg/kg rLZ-8 groups was signi cantly higher (P < 0.05), compared with the 56 μg/kg rLZ-8 group, the 112 μg/kg rLZ-8 group was signi cantly 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 signi cantly different in the other experimental groups, compared with in the model group,the 56 and 224 μg/kg rLZ-8 groups was signi cantly lower (P < 0.05), compared with the 56 μg/kg rLZ-8 group,the 112 μg/kg rLZ-8 groups was signi cantly higher (P < 0.05), compared with the 112 μg/kg rLZ-8 group,the 224 μg/kg rLZ-8 groups was signi cantly lower (P < 0.01), As shown in Table 16.
Compared with the normal saline group, the content of BDNF in the cerebral cortex of rats in the model group was signi cantly lower (P < 0.01) and the 112 μg/kg rLZ-8 group was signi cantly higher (P < 0.01); Compared with the model group, the donepezil and 112 μg/kg rLZ-8 groups was signi cantly increased (P < 0.01), and the224 μg/kg rLZ-8 group was also signi cantly 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 signi cantly decreased (P < 0.01). As shown in Table 16    scopolamine hydrobromide for one week, its effects on the organ indexes of rats were not signi cant, 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.

Discussion
Ganoderma lucidum, a traditional Chinese medicine, can improve the oxygen supply ability of hemoglobin, reduce the consumption of invalid oxygen, accelerate the blood circulation, and improve the oxygen supply and nutritional status of the brain, delaying the aging of the brain and showing a certain preventive effect on Alzheimer's disease. Fungal Immunomodulatory Protein of Ganoderma lucidium ( LZ-8), an active component of Ganoderma lucidum, was obtained by genetic engineering in our previous studies. In this study, the protective effects of recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) against the scopolamine-induced Alzheimer's disease in rats were studied.
Water maze was designed by Morris, a British cardiologist, in the early 1980s for observing the spatial learning and memory ability of experimental animals [18] . The results in this study showed that rLZ-8 and donepezil could signi cantly improve the learning and memory impairment induced by scopolamine hydrobromide, and the improvement of 56 µg/kg rLZ-8 on the learning and memory was better than that of the other doses of rLZ-8. Learning and memory is closely associated with the cholinergic system in basal forebrain (BF) and the content of acetylcholine in the brain tissue decreases with age.
Acetylcholinesterase is a key enzyme to hydrolyze acetylcholine, indirectly re ecting the content of acetylcholine, choline acetyltransferase is a key enzyme that coacts with acetylcholinesterase to regulate the content of acetylcholine in the central nervous system, and acetylcholinesterase together regulate the content of acetylcholine in the body. The results in this study were consistent with those reported by other scholars [18,19] . In this study, We found that the activities of acetylcholinesterase and choline acetyltransferase were associated with the dose of rLZ-8, and a low dose of rLZ-8 could signi cantly improve the learning and memory impairment induced by scopolamine hydrobromide. Free radical damage is the pathological basis of many diseases, and induced mainly by the uncontrollable and abnormal free radical reaction in cells, resulting in serious damages to the structure and function of cells, and free radicals are toxic products that are produced by organisms and can damage themselves, causing lipid peroxidation to produce lipid peroxides. The results in this study showed that the oxygen free radicals were scavenged to some extent in rats in the different rLZ-8-treated groups. NO (nitric oxide) as a vasoactive factor and a special neurotransmitter in the brain is involved in regulating the physiological process of cerebral blood ow, and learning and memory.
The contents of total TNOS andiNOS were detected in this experiment, and the results showed that the different doses of rLZ-8 could regulate the activity of NOS enzyme and improve the learning and memory of rats with Alzheimer's disease.
Some progress has been made in the study on the pathogenesis of in ammatory injury in AD in recent years [20][21][22] . The deposition of AP (amyloid protein) in the brain of AD patients can lead to the excessive secretion of in ammatory cytokines, complement and immune factors by glial cells, thus causing a strong neurotoxic effect. A large number of studies have found that there is a serious focal in ammatory reaction in the brain of patients with AD, in which pre-in ammatory factors such as IL-1β, IL-6 and TNF-a are closely related to the pathogenesis and progression of AD, and TNF-α is the earliest and important in ammatory mediator in the process of in ammatory reaction to activate neutrophils and lymphocytes, increase the permeability of vascular endothelial cells, regulate the metabolism of other tissues, and promote the synthesis and release of other cytokines [23,24] . IL-6 can induce the differentiation of B cells to produce antibodies, as well as the activation, proliferation and differentiation of T cells to participate in the immune response of the body, which is a provocative agent of in ammatory reaction. IL-1, also known as lymphocyte-stimulating factor, is mainly produced by activated monocytes-macrophages, and exists in the forms of IL-1α and IL-1β. The in ammation caused by the interaction of in ammatory factors is also one of the causes of Alzheimer's disease. The increased expression of in ammatory factors can activate microglia to engulf foreign bodies so as to improve the symptoms of Alzheimer's disease. In this study, the expression of in ammatory cytokines IL-1β, IL-6 and TNF-α in the hippocampus and cerebral cortex was affected by the different doses of rLZ-8. GDNF is a neurotrophic factor discovered in 1993, the target of GDNF is not only dopaminergic neurons in substantia nigra, but also cholinergic neurons in basal forebrain, and GDNF can reduce the dependence of basal cholinergic neurons on nerve growth factor and is closely related to many degenerative diseases of the nervous system, such as amyotrophic lateral sclerosis (AIS) and AD [25,26] . BDNF is one of the members of neurotrophic family [27] , widely distributed in the central nervous system, sensory nerve and spinal cord neurons, and is an important neurotrophic factor in neurons and glia; BDNF is closely associated with learning and memory, and the expression of BDNF in the hippocampus plays an important role in strengthening the memory process and long-term potentiation [28,29] . The decrease of BDNF in AD can weaken the function of hippocampus in two ways: one is the plasticity of synapses, that is, the de ciency of BDNF can weaken the ability of synapses to encode information and block the long-term potentiation, and the other is that the decrease of BDNF makes hippocampal neurons more vulnerable to damage and degeneration from the point of view of neurotrophic factor, mainly affecting the cholinergic system of basal forebrain. In this experiment, the different doses of rLZ-8 showed a certain effect on the expression of GDNF and BDNF in the hippocampus and cerebral cortex of rats with AD.

Limitations
In this study, we used scopolamine hydrobromide to induce AD rat model. Scopolamine hydrobromide can bind to choline receptor and block the binding of acetylcholine receptor, resulting in the decrease of acetylcholine transmitter level and the decrease of learning ability. However, the AD rat model induced by scopolamine hydrobromide is a transient model, and it lacks typical pathological changes such as degeneration of ad neurons and deposition of a β. In this experiment, the learning and memory ability of rats was evaluated by water maze test. The changes of cholinergic system level, antioxidant level, lipid peroxidation level and proin ammatory cytokines in serum, hippocampus and cortical tissue were detected by using the kit. The results showed that rlz-8 could improve the learning ability of transient AD rat model, but not from the water of signal pathway Objective to study the protective mechanism of rlz-8 on scopolamine induced AD rats. In the next step, we have established a rat model of ad induced by D-galactose. On the basis of previous studies, we intend to study the effect of rlz-8 on AD rat model from the level of pathology and signal pathway.

Conclusion
The above results indicate that rLZ-8 has some advantages over donepazil, because the immunomodulatory protein with less toxic and side effects on human body is different from donepezil, and moreover, rLZ-8 can be used for the prevention of learning and memory impairment for a long time, while donepezil can be only used for the treatment of mild to moderate learning and memory impairment, and can , t be used for the prevention of learning and memory impairment.
It was found in our study that rLZ-8 showed some effects on the content or expression of acetylcholinesterase, choline acetyltransferase, various oxygen free radicals, NO, IL-1β, IL-6, TNF-α, and GDNF in the serum and brain tissues of scopolamine-induced AD rats. The change of content or expression of BDNF in serum or brain tissue has certain effect. The mechanism of the protective effect of rLZ-8 against the scopolamine-induced AD in rats is to be investigated at the signal pathway level in our further study.rLZ-8 had no effect on the body weight of rats, but a signi cant effect on the organ indexes, indirectly suggesting that rLZ-8 had no signi cant toxic and side effect on rats with AD induced by scopolamine. RLZ-8 could signi cantly improve the learning and memory impairment of AD rats induced by scopolamine. In addition, rLZ-8 could improve the content or activity of related indexes, which may provide evidences for the results of behavioral experiments, and a basis for the study of rLZ-8 as a drug for the treatment of AD.  Effects of rLZ-8 on the body weight rats.