Application of PINK1 / OPTN / NDP52 pathway in improving mitochondrial autophagy in Alzheimer's disease

Abstract

The application of PINK1 / OPTN / NDP52 pathway to improve mitochondrial autophagy in Alzheimer's disease involves the field of disease treatment for Alzheimer's disease. First, in animal experiments, APP / PS1 transgenic mice were used to intervene with the traditional Chinese medicine Zhinaoshen to detect changes in the learning and memory abilities of mice and observe changes in the morphology of hippocampal neurons. 25‑35 After treatment, undifferentiated PC12 cells were observed for changes in cell morphology. Both experiments detected oxidative damage to neurons, mitochondrial autophagy, and mitochondrial function-related proteins to clarify the effects of drugs on the inhibition of mitochondrial autophagy disorders in AD model mice, thereby exploring and broadening the therapeutic drugs for Alzheimer's disease. Therefore, drugs that upregulate the PINK1 / OPTN / NDP52 pathway to improve mitochondrial autophagy in cells can be used in the preparation of therapeutic drugs for Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of disease treatment for Alzheimer's disease, and specifically to an application of a PINK1 / OPTN / NDP52 pathway in improving cell mitochondrial autophagy in Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, and its main clinical manifestations are progressive degeneration of understanding, memory, language expression, and cognition.

[0003] The pathological manifestations of AD are severe cortical atrophy, senile plaques (SP) with β-amyloid protein (Aβ) as the core component, neurofibrillary tangles (NFTs) caused by hyperphosphorylation of microtubule-associated τ protein (Tau), and neuropil threads.

[0004] At present, the clinical treatment of AD mainly improves patients' cognitive impairment through cholinesterase inhibitors (ChEI) and glutamate receptor antagonists, which are "symptomatic drugs" rather than "therapeutic drugs" for AD and cannot improve neurological lesions. Drugs such as β-secretase inhibitors and valproic acid that act to reduce the level of Aβ, inhibit the aggregation, proliferation and expression of Tau proteins, and target the stabilization of microtubules to improve the abnormal aggregation of Aβ and Tau proteins are still in the clinical research stage. Therefore, further exploration of the pathological mechanism of AD based on the pathological changes of Aβ and Tau proteins is not only the need for biomarkers in the clinical diagnosis of AD, but also the key to the early diagnosis and treatment of AD.

[0005] Mitochondrial dysfunction is a common pathological feature of transgenic animal models and human AD patients. Mitochondrial autophagy disorder is an important pathological change of AD. Under normal circumstances, damaged mitochondria often reduce damage to surrounding organelles and the entire cell through mitophagy, that is, damaged mitochondria are engulfed and cleared by lysosomes after binding to autophagosomes. When mitophagy is abnormal, damaged mitochondria will accumulate, leading to a variety of cellular changes including microRNA dysregulation, cell respiratory failure under reactive oxygen species (ROS) stimulation, and synaptic dysfunction. Mitochondrial autophagy is related to both before and after the pathology of Aβ and Tau proteins: in vitro neuronal culture suggests that mitochondrial dysfunction can promote the production of amyloid proteins through oxidative stress, and after the pathology of Aβ and Tau proteins, animal experiments have found that the deposition of Aβ can cause abnormal mitochondrial function and synaptic activity in small hippocampal cells, impairing mitochondrial autophagy.

[0006] Previous studies have found that the Chinese medicine Zhinaoshen, which replenishes qi and activates blood circulation, can promote the generation of new mature neurons (BrdU+-MAP-2+) on the ischemic side, inhibit the expression of the autophagy gene Beclin-1, inhibit the autophagy of neurons in the ischemic peripheral area, promote neurogenesis, and ultimately reduce neuronal damage caused by ischemia-reperfusion. However, there is still a lack of in-depth research on its mechanism of inhibiting neuronal autophagy. Summary of the invention

[0007] The present invention provides a new theoretical basis and idea for treating AD by improving mitochondrial autophagy with traditional Chinese medicine from two parts of experiments, animal experiment and cell experiment.

[0008] In animal experiments, APP / PS1 transgenic mice were used to intervene with the traditional Chinese medicine Zhinaoshen to detect changes in the learning and memory abilities of mice and observe changes in the morphology of hippocampal neurons. Cell experiments used Aβ 25-35 The undifferentiated PC12 cells after treatment were observed for changes in cell morphology. Both parts of the experiment detected oxidative damage to neurons, mitochondrial autophagy, and proteins related to mitochondrial function, so as to clarify the effects of drugs that inhibit mitochondrial autophagy disorders in AD model mice, thereby exploring and broadening the therapeutic drugs for Alzheimer's disease.

[0009] Both parts of the experiment observed the morphological changes of neurons, detected oxidative damage of neurons, mitochondrial autophagy and proteins related to mitochondrial function, so as to clarify the effects of Chinese medicine for invigorating qi and activating blood circulation on mitochondrial autophagy disorders in AD model mice.

[0010] Animal experiments have found that Chinese medicine for invigorating qi and activating blood circulation can improve the learning and memory ability of APP / PS1 mice, improve the morphology of hippocampal tissue cells, upregulate the expression of PINK1, OPTN, NDP52, LAMP1, PDS59 and LC3 proteins, and increase the expression of Beclin and BCL2 proteins; cell experiments have found that Chinese medicine serum for invigorating qi and activating blood circulation can improve the survival rate of undifferentiated PC12 cells, increase mitochondrial membrane potential, increase the expression of mitochondrial PINK1, OPTN, NDP52, LC3, LAMP1 proteins, and increase the content of Beclin and BCL2 proteins, indicating that Chinese medicine for invigorating qi and activating blood circulation can simultaneously increase the total expression of autophagy-related proteins in hippocampal tissue and mitochondria, improve cell mitochondrial autophagy by upregulating the PINK1 / OPTN / NDP52 pathway, increase mitochondrial membrane potential, and reduce cell oxidative damage, thereby improving the morphology of nerve cells and increasing cell survival rate.

[0011] Therefore, the present invention proposes an application of the PINK1 / OPTN / NDP52 pathway to improve mitochondrial autophagy in Alzheimer's disease, specifically: a drug that upregulates the PINK1 / OPTN / NDP52 pathway to improve mitochondrial autophagy can be used in the preparation of a drug for treating Alzheimer's disease.

[0012] According to the experimental results of the present invention, the survival rate of undifferentiated PC12 cells can be improved, the mitochondrial membrane potential can be improved, the expression of mitochondrial PINK1, OPTN, NDP52, LC3, and LAMP1 proteins can be increased, and the content of Beclin and BCL2 proteins can be increased. At the same time, the drug that can increase the total expression of autophagy-related proteins in hippocampal tissue and mitochondria can improve cell mitochondrial autophagy by upregulating the PINK1 / OPTN / NDP52 pathway, increase mitochondrial membrane potential, and reduce cell oxidative damage, thereby improving the morphology of nerve cells, increasing cell survival rate, and achieving the treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 To study the effect of the Qi-invigorating and blood-activating method on the water maze escape latency of APP / PS1 double transgenic mice.

[0014] Figure 2 The effect of the method of replenishing qi and activating blood circulation on the pathological morphology of brain tissue in APP / PS1 double transgenic mice. Note: A, C57 control group; B, APP / PS1 model group; C, APP / PS1+replenishing qi, activating blood circulation and improving intelligence Chinese medicine group; D, APP / PS1+memantine group.

[0015] Figure 3 To investigate the effect of the Qi-invigorating and blood-activating method on the expression of mitochondrial-related proteins in APP / PS1 model mice.

[0016] Figure 4 Aβ 25-35Effects on the morphology of undifferentiated PC12 cells (HE×400). Note: A, cell slides of the control group; B, cell slides of the model group; C, cell images of the control group; D, cell smears of the model group.

[0017] Figure 5 is the ROS content of PC12 cells in each group. Note: ①, control group; ②, model group; ③, untreated ROS negative control group; ④, ROS positive control group.

[0018] Figure 6 Chinese medicine for invigorating qi and promoting blood circulation has an effect on Aβ 25-35 Effect of injury on the expression of mitochondrial autophagy-related proteins in PC12 cells.

[0019] Figure 7 Chinese medicine for invigorating qi and promoting blood circulation has an effect on Aβ 25-35 Effect of injury on the expression of Beclin and Bcl2 proteins in PC12 cells. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with embodiments and drawings.

[0021] The research proposed in this invention is divided into two parts:

[0022] Experiment 1: Study on the protective mechanism of Zhinao Shen on aged and APP / PS1 transgenic mice. The AD mouse model was constructed by APP / PS1 transgenic mice, and the changes in learning and memory ability of the double transgenic model mice after medication and the changes in cell morphology of the hippocampal tissue of each group of mice were observed. The effects of traditional Chinese medicine on the mitochondrial autophagy function of the hippocampal tissue of AD model mice were analyzed by detecting the related indicators of oxidative damage of hippocampal tissue cells and the changes in the main proteins of the mitochondrial autophagy pathway.

[0023] Experiment 2: Effect of Zhinao Ginseng on Aβ in vitro 25-35 Study on PC12 cells damaged by Aβ 25-35 Undifferentiated PC12 cells were treated to construct AD model cells, and the survival rate and ROS content of the model cells were detected, and the cell HE staining smears were observed. After grouping treatment, the cell survival rate, mitochondrial membrane potential, intracellular ROS content, mitochondrial autophagy-related proteins (LC3Ⅰ / Ⅱ, PINK1, NDP52, OPTN, mitochondrial membrane protein LAMP1, cathepsin D, hippocampal synaptic protein PSD-95, Beclin 1 protein, anti-apoptotic protein Bcl-2), cell apoptosis-related proteins (Beclin protein, Bcl2 protein) and cell function-related proteins (Cyt-C, MRCCⅡ, SOD2, ATP) were compared among the groups.

[0024] It is further proved that Chinese medicine protects neurons by affecting the mitochondrial autophagy function of AD model cells and improving the pathological damage of AD model cells.

[0025] Example 1

[0026] Study on the protective mechanism of Zhinaoshen, a traditional Chinese medicine for invigorating qi and promoting blood circulation, on aged and APP / PS1 transgenic mice

[0027] Eighteen 25-month-old C57BL mice and 54 2-month-old APP / PS1 double transgenic mice were purchased from the Animal Research Center of the Chinese Academy of Sciences, half of which were male and half were female.

[0028] Experimental model preparation: APP / PS1 double transgenic mice were used in the AD model group.

[0029] Experimental groups: ①C57BL mice (control group); ②APP / PS1 model group (model group); ③APP / PS1 model + memantine group (Western medicine group); ④APP / PS1 model group + Zhinaoshen, a traditional Chinese medicine for invigorating qi and promoting blood circulation (traditional Chinese medicine group)

[0030] The drug was administered by intragastric gavage in groups for 60 days. The details are as follows:

[0031] ①C57BL control group: 10 mL kg -1 Body weight was measured by intragastric administration of normal saline once a day between 8 and 9 am.

[0032] ②APP / PS1 model group: 10 mL kg -1 Body weight was administered by intragastric administration of normal saline once a day.

[0033] ③ APP / PS1+Qi-invigorating, blood-activating and intelligence-enhancing Chinese medicine group: 10 mL·kg -1 Weight-based Chinese medicine for invigorating qi, activating blood circulation and improving intelligence was administered orally once a day. Chinese medicine for invigorating qi, activating blood circulation and improving intelligence (composed of ginsenosides, crocins and protocatechuic acid from ginsenosides), which contained 22.9% ginsenosides, 33.8% protocatechuic acid from ginsenosides and 4.5% crocins. The single drug dose was 33.0 mg kg -1 .

[0034] ④ APP / PS1+memantine group: 10 mL kg -1 Memantine aqueous solution was administered orally based on body weight, once a day.

[0035] 1.1 Behavioral testing of each group

[0036] 1.1.1 Morris water maze test

[0037] Morris water maze (Model 1056001, Beijing Zhongshidichuang Technology Development Co., Ltd.) divides the pool into 4 quadrants, and the platform is placed in the middle of the fourth quadrant, with a diameter of 9 cm and a height of 18 cm. Add water to the pool, the water depth is about 20 cm, the water surface is 0.5 cm above the platform surface, the water temperature is 25±1℃, and skimmed milk powder is injected and mixed to make the water opaque milky white, so that the animal vision cannot distinguish whether there is a platform in the pool, and the computer can successfully track the activities of the mice. The experiment was carried out in a soundproof room. Various experimental conditions such as the position of the pool, the position and intensity of the light source remained unchanged throughout the experiment to determine the position of the mouse's positioning platform. The test procedures include: ① Positioning navigation experiment: used to measure the learning and memory ability of animals in the water maze. It is carried out once a day and the training is continuous for 4 days. Training method: During the experiment, rats were trained twice a day at a fixed time period. Two different water entry points were randomly selected (following the random principle). The mice were placed in the water facing the wall. The automatic video system recorded the time required for the mice to find and climb onto the platform from entering the water (based on the four limbs climbing onto the platform), that is, the escape latency, and the swimming distance. If the mouse did not find the platform within 3 minutes, the experimenter guided it to the platform and stayed for -5-10 seconds. The escape latency was recorded as 90 seconds. ② Spatial exploration experiment: used to measure the animal's accurate memory of the spatial location of the platform, that is, the memory retention ability. On the afternoon of the 4th day, the platform was removed, and two water entry points were randomly selected. The mice were placed in the pool facing the pool wall. The camera system automatically recorded the number of times the mice entered and exited the original platform area within 3 minutes and the proportion of the time they stayed in the fourth quadrant where the original platform was located as an indicator for evaluating the learning and memory ability of the mice.

[0038] like Figure 1 As shown in Table 1, during the 4-day training period, the escape latency of mice in each group decreased with the increase of training days. The escape latency of mice in the control group and the Chinese medicine group was significantly lower than that in the model group; on the 3rd and 4th days of training, the escape latency of the Chinese medicine group was significantly lower than that of the Western medicine group.

[0039] Table 1: Results of water maze navigation experiment

[0040]

[0041] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01; compared with the western medicine group: △ P < 0.05; △△ P<0.01

[0042] Table 2 shows that after the training, the number of times the control group and the Chinese medicine group crossed the platform increased compared with the model group and the Western medicine group, which was statistically significant; the proportion of the time the control group and the Chinese medicine group stayed in the platform target quadrant (quadrant 4) increased compared with the model group and the Western medicine group, which was statistically significant.

[0043] Table 2: Results of water maze spatial exploration experiment

[0044]

[0045] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01; compared with the western medicine group: △ P < 0.05; △△ P<0.01

[0046] 1.1.2 Jumping platform test

[0047] The jumping platform device (model 1049002, Beijing Zhongshidichuang Technology Development Co., Ltd.) is a plexiglass box with a length of about 120 cm, a width of 12 cm, and a height of 30 cm. It is divided into 4 chambers, which can allow 4 mice to be tested at the same time. The bottom of the experimental box is covered with a copper grid, which is connected to a power supply and the current is controlled at 36 V. An insulating platform (each chamber is 12×12×30 cm long) is placed on the copper grid in the experimental box near a corner of the box as a safe area for animals to avoid electric shock. The experimental device is connected to a computer automatic recording system. During the experiment, the mice were placed in the jumping platform experimental box and adapted to the environment for 5 minutes. After that, the mice were gently placed on the jumping platform and the copper grid was electrified. When the mice jumped off the jumping platform and their limbs touched the copper grid, they would be shocked. The normal avoidance reaction was to jump onto the jumping platform and return to the safe area to avoid electric shock. The learning was carried out for 5 minutes, and the number of electric shocks (number of errors) within 5 minutes was recorded as the learning results. Memory ability was tested 24 hours later. The mice were placed on a jumping platform and the latency period, the time from when the mice stayed on the platform to when they jumped off for the first time and received an electric shock, and the number of errors within 5 minutes (the number of times the mouse's four limbs simultaneously touched the copper grid and were shocked) were recorded as evaluation indicators of memory function.

[0048] Table 3 shows that the platform jumping latency in the control group was higher than that in the model group and the western medicine group, and the platform jumping latency in the traditional Chinese medicine group was higher than that in the model group, and the differences were statistically significant; the number of errors in the control group was lower than that in the model group and the western medicine group, and the number of errors in the traditional Chinese medicine group was lower than that in the model group and the western medicine group, and the differences were statistically significant.

[0049] Table 3: Jumping platform test results

[0050]

[0051] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01; compared with the western medicine group: △ P < 0.05; △△ P<0.01

[0052] 1.2 Pathological examination of each group

[0053] 1.1 Electron microscopic observation of pathological morphology

[0054] The morphology of mouse hippocampal cells was observed by transmission electron microscopy. Figure 2 As shown in the figure, the C57 control group showed that the hippocampal neuron structure was intact, the pyramidal neuron nuclei were normal in morphology and evenly distributed, there was a large and round nucleus in the center of the pyramidal neuron nucleus, the nucleolus was large and obvious, the cytoplasm was rich, there was no dissolution or condensation, the nuclear membrane was clear, very few glial cells were visible, and no abnormal changes in glial cells were observed. APP / PS1 model group: the number of neurons decreased, and necrosis and degeneration occurred. The pyramidal cells were reduced in size, the cell bands were disordered and incomplete, scattered, irregular in shape, the nuclei were condensed, and glial cell proliferation occurred.

[0055] APP / PS1+Memantine group: Pyramidal cells were arranged relatively neatly, with a relatively uniform distribution. The intracellular structure was stained normally, with the cell body and cytoplasm light blue. The cell cytoplasm staining area was reduced. There were a small number of pyknotic necrotic neurons, and the cell membrane and nuclear membrane boundaries were relatively clear. APP / PS1+Yiqi Huoxue Yizhi Chinese medicine group: Pyramidal cells were arranged neatly, with complete cell layers, normal morphology, dense structure, and relatively uniform distribution. The intracellular structure was stained normally, with the cell body and cytoplasm light blue, large and obvious nucleoli, abundant cytoplasm, and clear nuclear membrane. A small number of glial cells were visible, and no abnormal changes were observed in glial cells.

[0056] 1.2 Detection of hippocampal related proteins in each group

[0057] The antioxidant indicators including superoxide dismutase (SOD) activity, cytochrome c oxidase I (COI), adenosine triphosphate (ATP) content, and reactive oxygen species (ROS) fluorescence intensity of the hippocampal tissue of each group of mice were detected, as well as apoptosis indicators including cysteine ​​protease 3 (Caspase3), cysteine ​​protease 8 (Caspase8), and cysteine ​​protease 9 (Caspase9): SOD activity was determined by colorimetry using a SOD kit (provided by Nanjing Jiancheng Bioengineering Institute, batch number: 20201115), and the change in optical density was measured at 405 nm; COX was determined by spectrophotometry using a COX kit (provided by Nanjing Jiancheng Bioengineering Institute, batch number: 20201019), the change in optical density was measured at 550 nm, and the activity of COX was calculated based on this; ATP content was determined by phosphomolybdic acid colorimetry using an ATP kit ( The change of optical density was measured at 700 nm, and the depth of color was proportional to the phosphate content; the DCFH-DA probe was used to detect the ROS fluorescence intensity, and the ROS kit (Weiglas Biotechnology (Beijing) Co., Ltd., batch number: 20201014) was used to obtain the total level of reactive oxygen in cells or tissues at an excitation wavelength of 485 nm and an emission wavelength of 538 nm; the corresponding ELISA kits were used for cysteine ​​proteases, and the enzyme-linked immunosorbent assay was used for detection, Caspase3 ELISA kit (Beijing Xinbosheng Biotechnology Co., Ltd., batch number: 20201115), Caspas8 ELISA kit (Beijing Xinbosheng Biotechnology Co., Ltd., batch number: 20201123), and Caspas9 ELISA kit (Beijing Xinbosheng Biotechnology Co., Ltd., batch number: 20201126). Determination instruments: DR-200BS, fully automatic enzyme label analyzer, produced by Wuxi Huawei Delang Instrument Co., Ltd.; ROCHE / E601, fully automatic electrochemiluminescence immunoassay analyzer (produced by ROCHE, Switzerland).

[0058] Table 4 shows that compared with the model group, the SOD protein and ROS fluorescence intensity of the control group decreased, and the intracellular ATP content and COI expression increased relatively, and the differences were statistically significant; compared with the model group, the SOD protein and ROS fluorescence intensity of the Chinese medicine group increased, and the ATP and COI expressions decreased, and the differences were statistically significant; in terms of SOD protein expression and ATP content, the Chinese medicine group increased compared with the Western medicine group, and the differences were statistically significant.

[0059] Table 4: Antioxidant-related proteins in mouse hippocampus tissue

[0060]

[0061] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01; compared with the western medicine group: △ P < 0.05; △△ P<0.01

[0062] Table 5 shows that the expression of apoptosis-related proteins Caspase3, Caspase8 and Caspase9 in the control group was lower than that in the model group, and the difference was statistically significant; the expression of Caspase3 in the Chinese medicine group and the Western medicine group was lower than that in the model group, and the expression of Caspase3, Caspase8 and Caspase9 in the Chinese medicine group was lower than that in the Western medicine group, and the difference was statistically significant.

[0063] Table 5: Apoptosis-related indicators of mouse hippocampal tissue

[0064]

[0065] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01; compared with the western medicine group: △ P < 0.05; △△ P<0.01

[0066] 1.3 Western Blot detection of hippocampal tissue in each group

[0067] Hippocampal tissues of 6 mice were randomly selected from each group to examine the expression of LC3I / II, PINK1, NDP52, OPTN, mitochondrial membrane protein LAMP1, CathepsinD, synaptic protein PSD-95, Beclin1 and Bcl-2 protein antibodies.

[0068] First, extract the protein from the brain tissue, pre-cool the RIPA protein extraction reagent, and add protease inhibitors (phosphorylated proteins need to be added with phosphatase inhibitors). Add 0.1MPMSF mother solution before protein extraction, with a final concentration of 1mM. Weigh the tissue weight and add the lysis solution at a ratio of weight: lysis solution volume = 1:9. Homogenize three times with an interval of 10s (15000rpm, 10s). After homogenization, incubate on ice for 20min, then centrifuge (13000rpm, 20min, 4 degrees). After centrifugation, take the supernatant, aliquot and store for testing.

[0069] For protein quantitative determination, prepare BCA working solution = 50:1, and dilute each extracted BSA standard. Dilute the sample with PBS. Add BCA working solution = 1:8 to the sample, mix well, incubate at 37 degrees for 30 minutes or at room temperature for 60 minutes, and read the OD value with a 570nm wavelength filter on the microplate reader. Then adjust the protein concentration with RIPA, and add 5 times the sample reduction buffer to a final sample concentration of 4mg / ml. Adjust the sample protein concentration.

[0070] For the WB formal experiment of the target protein, according to the molecular weight of the target protein, 12% and 8% separation gels were prepared, the concentration of the concentrated gel was 5%, and the protein sample to be tested was loaded (20ug / well, the concentrated gel constant voltage was 90V20min, the separation gel constant voltage was 160V). Electrophoresis, wet transfer (300mA constant current, 0.45um pore size NC membrane, transfer time 1-2h), after the transfer was completed, the transfer effect was observed by staining with Ponceau red staining reagent; blocking: the membrane was completely immersed in 3% BSA-TBST and gently shaken at room temperature for 30min.

[0071] Primary antibody incubation: dilute the primary antibody with 3% BSA-TBST, incubate at room temperature for 10 minutes, and place at 4°C overnight. The primary antibodies include LC3I / II, PINK1, NDP52, OPTN, mitochondrial membrane protein LAMP1, CathepsinD, synaptic protein PSD-95, Beclin1 and Bcl-2 protein. After incubation at room temperature for 30 minutes, wash the membrane (wash the membrane 5 times with TBST, 3 minutes each time); Secondary antibody incubation: dilute the secondary antibody with 5% skim milk powder-TBST, goat anti-rabbit IgG (H+L) HRP, 1:10000, goat anti-mouse IgG (H+L) HRP, 1:10000, shake gently at room temperature for 40 minutes, and then wash the membrane (wash the membrane 6 times with TBST, 3 minutes each time). After adding ECL to the membrane, react for 3-5 minutes, film exposure: 10s-5min (exposure time is adjusted according to different light intensities), develop for 2 minutes, and fix.

[0072] The formal experiment of WB of internal reference protein is as follows: Wash the membrane with Stripping Buffer at 37℃ for 30min (if the molecular weight difference between the target protein and the internal reference protein is more than 10K, the step of washing the membrane with stripping buffer can be omitted). Wash the membrane 3 times with deionized water, and then wash the membrane 3 times with TBST, each time for 3min. Immerse the membrane completely in 5% skim milk powder-TBST and shake gently at room temperature for 30min. Incubation internal control: add β-actin mouse monoclonal antibody, dilute the antibody with 5% skim milk powder-TBST, dilute 1:5000, incubate at room temperature for 40 minutes; wash the membrane (wash the membrane with TBST for 5 times, 3 minutes each time) and then incubate with secondary antibody: dilute the secondary antibody with 5% skim milk powder-TBST, goat anti-mouse IgG (H+L) HRP, 1:10000, and shake gently at room temperature for 40 minutes; wash the membrane (wash the membrane with TBST for 5 times, 3 minutes each time), add ECL to the membrane and react for 3-5 minutes, film exposure: 10s-5min (exposure time is adjusted according to different light intensities), develop for 2min, and fix.

[0073] The results showed that compared with the control group, the expression of PINK1, OPTN, NDP52, PSD59, Beclin, and BCL2 proteins in the model group and the western medicine group decreased, while the expression of PINK1, OPTN, NDP52, PSD59, Beclin, and BCL2 proteins in the traditional Chinese medicine group increased compared with the model group, and the differences were statistically significant ( Figure 3 and Table 6).

[0074] Table 6: Mouse mitochondrial autophagy-related proteins

[0075]

[0076] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01; compared with the western medicine group: △ P < 0.05; △△ P<0.01

[0077] Example 2

[0078] Effect of Zhinao Shen on Aβ in vitro 23-35 Study on damaged PC12 cells

[0079] The experiment used undifferentiated semi-suspension PC12 cells donated by the Institute of Geriatrics, Capital Medical University. They were cultured in a constant temperature incubator at 37°C, 5% CO2 and saturated humidity in the corresponding culture medium. The medium was changed every 2 to 3 days, and the cells were subcultured when they grew to 90%.

[0080] Experimental model preparation: P12 cells were divided into a control group and a model group. The two groups of cells were cultured at a rate of 5×10 4 The cells were plated at a density of 100 μL / mL in a 96-well plate, 200 μL / well, and Aβ was added according to the group after culturing for 24 h. 25-35 The cells in the two groups were cultured at 20umol / L. The survival rate and ROS content were measured by CCK-8 method and the HE staining smears were observed to determine whether the model preparation was successful.

[0081] Experimental groups: ① Control group: After 24 hours of culture, control serum was added to PC12 cells, and then cultured for another 24 hours. ② Model group: PC12 cells were added with 20umol / L-Aβ 25-35 After 24 hours of culture, control serum was added and cultured for another 24 hours. ③ Zhinao Shen group: PC12 cells were added with 20umol / L-Aβ 25-35 After 24 hours of culture, the Zhinaoshen compound drug-containing serum was added for 24 hours.

[0082] The experiment was divided into three parts: ① Aβ 25-35 Preparation and testing of damaged cell models; ② Effects of serum concentration of Zhinaoshen, a traditional Chinese medicine for invigorating qi and promoting blood circulation, on the survival rate of undifferentiated PC12 cells; ③ Effects of serum of Zhinaoshen, a traditional Chinese medicine for invigorating qi and promoting blood circulation, on the survival rate of undifferentiated PC12 cells 25-35 Effects of damaged PC12 cells.

[0083] 2.1Aβ 25-35 Injured cell model testing

[0084] 2.1.1Aβ 25-35 Preparation of damaged cell model

[0085] PC12 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin) at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2 to 3 days, and cells were passaged when they grew to 90%. 4 / mL, 200μL / well) and cultured for 24h (37℃, 5% CO2 box) and Aβ was added according to the group 25-35 20umol / L and then continue to culture for 24h; the cells were grouped as follows: ① PC12 cells + control serum for 24h (control group); ① PC12 cells + Aβ25-35 20umol / L + control serum for 24h (model group)

[0086] 2.1.2 Observation of pathological morphology by HE staining

[0087] HE staining can reveal the general morphological and structural characteristics of various tissues or cells. HE staining was performed on cell slides and cell smears of the two groups of PC12 cells, respectively, and images were collected under a 400x field of view. Cell slides showed that the PC12 cells in the control group had a clear structure, intact nuclei, more cell surfaces, and roughly spindle-shaped cell morphology; the PC12 cells in the model group grew densely, the nuclei were crowded and aggregated into clusters, the nuclei were of different morphologies, and the nuclear fragmentation and basophilic staining were less than those in the control group. Cell smears showed that the number of PC12 cells in the control group was large, the nuclear structure was complete and round, and the cells were tightly connected by axons; the number of PC12 cells in the model group was significantly reduced, and it was seen that the nuclear condensation, the connection between cells was loose, and the eosinophilic staining was reduced compared with the control group ( Figure 4 shown).

[0088] 2.1.3 CCK-8 method for detecting Aβ 25-35 Damaged cell survival rate

[0089] CCK-8 detection: After 24 hours of drug intervention, the 96-well plate was taken out, the culture medium was removed, 100 μL CCK-8 mixture was added to each well and incubated for 2 hours (37°C, 5% CO2 box), and then the absorbance at 450nm was measured by microplate reader. The results showed that the survival rate of PC12 cell model group (88.59±5.57%) was reduced compared with the control group (100±5.83%) (P=0.006<0.01).

[0090] 2.1.4 Detection of Aβ by flow cytometry 25-35 ROS content in damaged cells

[0091] DCFH-DA was diluted to 10 μM with serum-free medium; the collected cell pellet was suspended in the diluted DCFH-DA and incubated for 30 min (37°C, 5% CO2 cell culture incubator, inverted and mixed every 3-5 min). In addition to the grouping, another group of controls was set up. The control group and the model group were added with diluted DCFH-DA, and another group was set up as a negative control group without treatment and a group as a positive control group; the well plate was removed and the cells were washed three times with serum-free medium; 200 μL PBS buffer was added to each well, and the flow cytometer was used for detection.

[0092] The test results showed that the ROS in the control group (70.9) increased compared with the model group ( Figure 5 shown).

[0093] 2.2 Effect of serum concentration of Rhizoma Cibotii on the survival rate of undifferentiated PC12 cells

[0094] PC12 cells were cultured in a PC-12 (undifferentiated) cell-specific culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin) in a constant temperature incubator (37°C, 5% CO2, saturated humidity), and the medium was changed every 2 to 3 days. When the cells grew to 90%, they were passaged again. The cells were plated in a 96-well plate (2×10 4 / mL, 200μL / well) and cultured for 24h (37℃, 5% CO2 box); the cells were grouped and treated separately: ① no serum was added (blank group); ② 2.5%, 5%, 10%, 15%, 20% drug-containing serum was added (drug-containing serum group); ③ 2.5%, 5%, 10%, 15%, 20% blank serum was added for treatment (blank serum group); after treatment, the cells were cultured for 24h (37℃, 5% CO2 box), and then the 96-well plate was taken out; the culture medium was aspirated, 100μL CCK-8 mixture was added to each well and incubated for 2h (37℃, 5% CO2 box); the cell viability was detected by using an enzyme marker to measure the absorbance at 450nm.

[0095] The results in Table 7 showed that the cell survival rate in the blank group was lower than that in the drug-containing serum group and the blank serum group at each serum concentration (P<0.01); in the drug-containing serum group, the cell survival rate at 10% serum concentration was higher than that at other concentrations, and at 2.5% serum concentration and 5% serum concentration, the cell survival rate increased with the increase of serum concentration, which was statistically significant; but the difference in cell survival rate between 20% serum concentration and 15% serum concentration was not statistically significant; in the blank serum group, the cell survival rate increased with the increase of serum concentration at serum concentrations of 2.5%, 5%, and 10%, which was statistically significant; but the difference in cell survival rate between 20% serum concentration and 15% serum concentration was not statistically significant; when the serum concentration was above 5%, the cell survival rate in the drug-containing serum group was higher than that in the blank serum group, and the difference was statistically significant.

[0096] Table 7: Effects of different serum concentrations on PC12 cell survival rate

[0097]

[0098] Note: Compared with 2.5% concentration in the same group: 1) P<0.05, 1)) P<0.01; compared with 5% concentration in the same group: 2) P<0.05, 2)) P<0.01; compared with 10% concentration in the same group: 3) P<0.05, 3)) P<0.01; compared with 15% concentration in the same group: 4) P<0.05, 4)) P<0.01; at the same concentration, the drug-containing serum group was compared with the blank serum group:△ P<0.05, △△ P<0.05

[0099] 2.3 Effect of serum of Zhinaoshen, a traditional Chinese medicine for invigorating qi and promoting blood circulation, on Aβ 25-35 Effects of damaged PC12 cells

[0100] 2.3.1 Cell culture

[0101] The culture conditions of undifferentiated PC12 cells were the same as those in 2.2. The cells were grouped as follows: ① PC12 cells + control serum for 24 h (control group); ② PC12 cells + Aβ 25-35 20umol / L+control serum for 24h (model group); ③Aβ 25-35 20umol / L24h+Zhinaogin compound drug-containing serum for 24h (TCM group), the treatment method is the same as 2.2.

[0102] 2.3.2 Effects of Chinese medicines for invigorating qi and promoting blood circulation on Aβ 25-35 Effects of damaged PC12 cells on mitochondrial membrane potential

[0103] Flow cytometry was used to detect the mitochondrial membrane potential of each group of cells: after grouping, the well plate was removed, the cells were collected, and 0.5 mL of complete culture was added to resuspend the cells; 0.5 mL of JC-1 working solution was added and incubated for 20 min (37°C, 5% CO2 box); after centrifugation (600g, 4°C, 4 min), the supernatant was removed and 1 mL of JC-1 buffer was added to wash twice; finally, the cells were resuspended with 1 mL of JC-1 buffer and tested on the machine.

[0104] Table 8 shows that the proportion of cells with normal mitochondrial membrane potential in the model group was lower than that in the control group, and the proportion of cells with decreased mitochondrial membrane potential was higher. However, the proportion of cells with normal mitochondrial membrane potential in the Chinese medicine group was higher than that in the model group, and the proportion of cells with decreased mitochondrial membrane potential was lower, which was statistically significant.

[0105] Table 8: Changes in membrane potential of PC12 cells

[0106]

[0107] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01

[0108] 2.3.3 Effects of Chinese medicines for invigorating qi and promoting blood circulation on Aβ 25-35 Effects of damaged PC12 cell function-related proteins

[0109] The intracellular ATP content, Cyt-C concentration, Cyt-C concentration and SOD2 concentration were detected respectively. ATP content detection: collect cells into centrifuge tubes, discard the supernatant, extract the volume of solution (500:1) according to the number of cells (104), ultrasonically disrupt (ice bath, power 200W, ultrasound 2s, stop 1s, total time 1min) and centrifuge (10000g, 4℃, 10min), take the supernatant to another EP tube, add 500μL of chloroform, shake and mix thoroughly, centrifuge (10000g, 4℃, 3min), take the supernatant and place it on ice for testing. After thorough mixing, immediately measure the absorbance value A1 at 340nm for 10s, put the 96-well UV plate in a 37℃ constant temperature incubator, and immediately measure its absorbance value A2 at 3min 10s. Calculate ΔA respectively 测定 =A2 测定 -A1 测定 , ΔA 标准 =A2 标准 -A1 标准 ATP content (μmol / 10 6 cell)=ΔA 测定 ÷(ΔA 标准 ÷C 标准 )×V 提取 ÷5=0.125×ΔA 测定 ÷ΔA 标准 . C 标准 : Standard solution concentration, 0.625 μmol / mL; V 提取 :The volume of extract added was 1mL; Determination of Cyt-C concentration, MRCCⅡ concentration and SOD2 concentration: ① Set up standard wells, blank wells and sample wells, and add 50μL of standard of different concentrations to each standard well; ② Add 50μL of the sample to be tested to the sample well, and add 50μL of specimen diluent to the blank well; ③ Add 100μL of detection antibody labeled with horseradish peroxidase (HRP) to each well, seal the reaction well with a sealing film, and incubate at 37℃ for 60min; ④ Wash the plate 5 times; ⑤ Add 100μL of substrate mixture to each well, seal the reaction well with a sealing film, and incubate at 37℃ for 15min away from light; ⑥ Add 50μL of stop solution to each well, and detect the OD value of each well at a wavelength of 450nm within 15min.

[0110] Table 9 shows that the ATP content and MRCCⅡ concentration in the model group were lower than those in the control group, and the Cyt-C concentration and SOD2 concentration were higher than those in the control group, which were statistically significant; compared with the model group, the Cyt-C concentration, MRCCⅡ concentration and SOD2 concentration in the Chinese medicine group were lower, and the MRCCⅡ concentration was higher, which was statistically significant.

[0111] Table 9: Expression of proteins related to oxidative damage in PC12 cells

[0112]

[0113] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01

[0114] 2.3.4 Western Blot Detection of the Effect of Qi-invigorating and Blood-activating Chinese Medicine on Aβ 25-35 Effect of damaged PC12 cells on mitochondrial function and apoptosis

[0115] First, the protein of brain tissue was extracted, and the protein concentration was measured after taking the sample supernatant. According to the concentration measurement results, 5× loading buffer and PBS were added to adjust the protein concentration to ensure the consistency of protein concentration between different groups. After preparing the electrophoresis gel, the processed samples were loaded. Electrophoresis, transfer (4℃ ice bath, 200mA, 90min, 0.45μm membrane, blocking (TBST blocked PVDF membrane, room temperature shaker, 2h),

[0116] For primary antibody incubation, dilute the corresponding primary antibody with TBST containing 2% BSA and incubate overnight at 4°C; wash off the excess primary antibody after the end (wash the membrane with TBST 3 times, 10 min / time; secondary antibody incubation: dilute the HRP-labeled secondary antibody with blocking solution and incubate on a shaker at room temperature for 1 hour. After the end, wash off the excess secondary antibody (wash the PVDF membrane with TBST 3 times, 10 min / time). Add ECL to the membrane and expose it using an automatic chemiluminescence image analysis system; after the exposure, wash the membrane, block it again, incubate it with internal reference, and expose it with secondary antibody. The process is the same as above. Finally, use Image J software to analyze the grayscale value. The primary antibody dilution ratio is: PINK1 1:800 dilution, NDP52 1:800 dilution, OPTN 1:4000 dilution, LAMP11:1000 dilution, cathepsin D 1:4000 dilution, PSD-951:4000 dilution, LC3 1:2000 dilution; internal reference protein dilution ratios are: GAPDH 1:5000 dilution, Cox IV 1:4000 dilution, and secondary antibody dilution ratios are all 1:5000.

[0117] Table 10 and Figure 6 The results showed that the expression of PINK1, OPTN, NDP52, LC3 and LAMP1 proteins in the model group was lower than that in the control group, while the expression of PSD-95 protein was relatively increased, which was statistically significant; compared with the model group, the expression of PINK1, OPTN, NDP52, LC3 and LAMP1 proteins in the Chinese medicine group was increased, while the expression of PSD-95 protein was decreased, which was statistically significant.

[0118] Table 10: Expression of mitochondrial autophagy-related proteins in PC12 cells

[0119]

[0120] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P < 0.01;

[0121] 2.3.5 Immunofluorescence (IF) detection of the effects of traditional Chinese medicine for invigorating qi and promoting blood circulation on Aβ 25-35 Effect of damaged PC12 cells on apoptosis

[0122] The three groups of cells were permeabilized by using 0.3% Triton X-100 to break the membrane for 10 minutes, and the membrane was washed (washed 3 times with PBS solution, 5 minutes each time); the sample blocking solution was added and incubated in a wet box at room temperature for 2 hours; the primary antibody was added: Beclin and Bcl2 were added at a ratio of 1:200, and the centrifuged serum was added and incubated in a wet box at 4°C overnight; the secondary antibody was added: after rewarming for 30 minutes, the membrane was washed (rinsed 3 times with PBS solution, 5 minutes each time), the secondary antibody working solution was added at a ratio of 1:200, and incubated at 37°C for 2 hours; DAPI was taken out in advance, thawed at room temperature, stained for 10 minutes, and the membrane was washed; the slices were dried (the slices were sliced ​​and dried in a 60°C oven); the anti-fluorescence quenching sealing agent was used to seal the slices, and the images were collected at 200 times and 400 times the field of view. The results are shown in Table 11.

[0123] Table 11: Expression of anti-apoptotic proteins in PC12 cells

[0124]

[0125]

[0126] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01

[0127] 2.3.6 Effects of Chinese medicines for invigorating qi and promoting blood circulation on Aβ 25-35 Effect of ROS content on damaged PC12 cells

[0128] Flow cytometry was used to detect the mean fluorescent intensity (MFI) of ROS in each group of cells: after completing the grouping process, the well plate was taken out and the cells were washed once with PBS; the diluted Red CMXRos staining working solution; incubate for 30 minutes (37°C, 5% CO2 cell culture incubator), mix by inversion every 3-5 minutes; set up a blank control in addition to the grouping; remove the well plate and wash the cells three times with fresh culture medium; add 200 μL PBS buffer to each well and detect using a flow cytometer.

[0129] Table 12 shows that the ROS positive rates in the control group and the Chinese medicine group were higher than those in the model group, and the average fluorescence intensity of ROS in the control group was higher than that in the model group, and the difference was statistically significant.

[0130] Table 12: ROS positive rate and mean fluorescence intensity of PC12 cells

[0131]

[0132] Note: Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group: * P < 0.05; ** P<0.01

[0133] Experimental conclusions of Examples 1 and 2 (Effects of Chinese medicine for invigorating qi and promoting blood circulation on APP / PS1 model mice and Aβ 25-35 Effect of damaged PC12 cells on mitochondrial autophagy):

[0134] In terms of animal experiments, both the water maze and the step-through test showed that Chinese medicine for invigorating qi and activating blood circulation can improve the learning and memory ability of APP / PS1 model mice; the structure of the pyramidal cells in the hippocampus tissue of the APP / PS1+yizhi Chinese medicine group was more intact than that of the model group, and no abnormal changes were found in the glial cells; compared with the model group, the SOD protein in the Chinese medicine group increased, and the ROS fluorescence intensity and COI protein expression decreased (P<0.05); the expression of Caspase3, Caspase8 and Caspase9 proteins decreased (P<0.05); the expression of mitochondrial-related proteins: the expression of PINK1, OPTN, NDP52, LAMP1, PDS59 and LC3 proteins in the Chinese medicine group increased, and the expression of apoptosis proteins Beclin and BCL2 proteins increased.

[0135] In terms of cell experiments, the Aβ was confirmed by observing the HE staining of cells (the number of PC12 cells in the model group decreased compared with the control group, the nucleus was condensed, and the surface protrusions were reduced), detecting the cell survival rate (the number of cells in the model group decreased) and the ROS content. 25-35The damaged cell model was successfully established; undifferentiated PC12 cells were treated with different concentrations of Qi-invigorating and blood-activating Chinese medicine serum and blank serum, and the cell survival rate was compared. It was found that the cell survival rate (%) was the highest (230.83±6.29) at a concentration of 10% serum, and when the serum concentration was above 5%, the cell survival rate of the serum-containing group was higher than that of the blank serum group (P<0.05), indicating that the Qi-invigorating and blood-activating Chinese medicine serum can improve the survival rate of undifferentiated PC12 cells; the cell model was constructed and grouped using the above modeling method. The percentage of cells with normal mitochondrial membrane potential in the Chinese medicine group increased compared with the model group, the expression of Cyt-C and SOD2 decreased, and the expression of MRCCⅡ increased; the expression of mitochondrial PINK1, OPTN, NDP52, LC3, and LAMP1 proteins increased, the expression of PSD-95 decreased, and the expression of Beclin and BCL2 proteins increased ( Figure 7 shown).

[0136] Therefore, Qi-invigorating and blood-activating Chinese medicine can improve cell mitochondrial autophagy and reduce cell apoptosis by regulating the PINK1 / OPTN / NDP52 pathway, thereby alleviating cell oxidative damage. This confirms that drugs that upregulate the PINK1 / OPTN / NDP52 pathway to improve cell mitochondrial autophagy can be used in the preparation of drugs for the treatment of Alzheimer's disease.

[0137] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. An application of PINK1 / OPTN / NDP52 pathway to improve mitochondrial autophagy in Alzheimer's disease, characterized in that: Drugs that upregulate the PINK1 / OPTN / NDP52 pathway to improve mitochondrial autophagy in cells can be used in the preparation of drugs for the treatment of Alzheimer's disease.

2. The use according to claim 1, characterized in that The drug can improve the survival rate of undifferentiated PC12 cells, increase mitochondrial membrane potential, increase the expression of mitochondrial PINK1, OPTN, NDP52, LC3, and LAMP1 proteins, and increase the content of Beclin and BCL2 proteins. At the same time, it can increase the total expression of autophagy-related proteins in hippocampal tissue and mitochondria. It can improve cell mitochondrial autophagy by upregulating the PINK1 / OPTN / NDP52 pathway, increase mitochondrial membrane potential, and reduce cell oxidative damage, thereby improving the morphology of nerve cells, increasing cell survival rate, and achieving the treatment of Alzheimer's disease.

3. The use according to claim 1 or 2, characterized in that: The active ingredients of the medicine are composed of ginseng, saffron and Alpinia oxyphylla.

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