Application of Erzhi pill extract in improving D-gal induced astrocyte energy metabolism disorder

Abstract

The invention discloses an effect of an Erzhi pill extract in improvement of D-gal induced astrocyte energy metabolism disorder. The method specifically comprises the following steps: inducing C8-D1A mouse astrocytes by using D-gal to establish an energy metabolism disorder model, screening out an optimal modeling condition, screening out the optimal administration concentration of the Erzhi pill extract by using an MTT experiment and an ATP kit, detecting the expression levels of glycogen synthase protein and glycogen synthase gene by using Western blot and RT-qPCR, and determining the energy metabolism disorder of the Erzhi pill extract according to the expression level of the Erzhi pill extract and the expression level of the Erzhi pill extract. The kit is used for detecting the levels of glucose-6-phosphoric acid, lactic acid and ATP (adenosine triphosphate) and the activity of hexokinase and glycogen phosphorylase a in cells, and the Erzhi pill extract can improve the glycogen metabolism of astrocytes, improve the generation of glycogenic lactic acid, further provide energy for the activity of neuronal cells and improve the energy metabolism disorder of D-gal induced astrocytes.

Description

Technical Field

[0001] This patent relates to the field of biomedicine technology, specifically to the research and application of neuropharmacology and neuroprotective mechanisms, especially the application of Erzhiwan extract in improving astrocyte energy metabolism disorders induced by D-gal. Background Art

[0002] Brain energy metabolism disorder refers to a pathological state in which the balance of energy production, supply and utilization in brain cells is broken, resulting in interference with normal physiological activities. It is particularly common in the study of neurodegenerative diseases such as Alzheimer's disease (AD). AD is a degenerative disease of the central nervous system with a complex pathogenesis. Previous studies on traditional pathogenesis such as Aβ deposition, Tau phosphorylation, neuroinflammation and oxidative stress have not obtained effective therapeutic targets and strategies. Due to the single energy source of the brain, it is extremely susceptible to energy metabolism disorders. Brain energy metabolism disorders and persistent brain energy gaps will appear in the early stage of AD, leading to dysfunction of neurons and astrocytes. Neurons are the ultimate beneficiaries of glucose uptake, and most glucose can only reach neurons after being transported by astrocytes. When neurons have high energy needs, astrocytes will respond and quickly break down their own glycogen to produce lactate for transport to neurons. Astrocytes play an important role in supporting and protecting neurons. Therefore, some scholars have proposed that preventing the occurrence and development of AD by improving, protecting or restoring brain energy status has become a new direction for the development of AD therapeutic drugs. Based on the previous findings of the research team, Erzhi Pills may improve learning and memory impairment in AD mice by regulating brain energy metabolism, and there are a large number of experimental studies on the Chinese medicine and its chemical components in the prevention and treatment of AD in terms of energy metabolism. The extract of Erzhi Pills contains monomer components such as salidroside, wedelolactone, luteolin, oleanolic acid, and privet glycosides. This patent aims to explore the role of phytoestrogen components in Erzhi Pills extracts in regulating energy metabolism through estrogen receptors, in order to provide new ideas and methods for the treatment of neurodegenerative diseases such as AD. Summary of the invention

[0003] In order to solve the technical problems mentioned in the background technology, based on the role of Erzhi Pills in regulating brain energy metabolism, the present invention finds that the use of Erzhi Pills extract can increase the glycogen content in astrocytes to varying degrees.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] 1. The energy metabolism disorder model was established by treating C8-D1A mouse astrocytes with D-galactose (D-gal) for different time periods (6 hours, 12 hours, 24 hours, 48 ​​hours) and different concentration gradients to determine the optimal modeling conditions. Subsequently, the improvement effect of Erzhiwan extract on the disease model was tested to find the optimal therapeutic concentration. Finally, the effect on brain glycogen levels was determined by a glycogen kit for subsequent experiments.

[0006] 2. By establishing a D-gal-induced astrocyte energy metabolism disorder model, a comparative study was conducted in multiple groups. The expression levels of proteins such as GPR30, PI3K, Akt, GSK3β, p-GSK3β and glycogen synthase were detected by Western blot technology, and the expression levels of corresponding genes were detected by RT-qPCR technology. In addition, the intracellular glucose-6-phosphate, lactate, ATP levels and the activities of hexokinase and glycogen phosphorylase a were determined using a kit to explore the effect of Erzhiwan extract on energy metabolism disorders through a certain signaling pathway.

[0007] The above scheme is used to explore the application of Erzhiwan extract in improving D-gal-induced astrocyte energy metabolism disorders and delaying the development of AD.

[0008] The present invention is based on the conclusion that the research team has previously proved that Erzhi Pills have a good therapeutic effect on AD from the overall efficacy level. On this basis, the molecular mechanism of the estrogen component of Erzhi Pills in treating AD is further elucidated; in vitro experiments are used to explore the molecular mechanism by which the estrogen component of Erzhi Pills plays a neuroprotective role in treating AD from the perspective of regulating energy metabolism disorders, providing an experimental and theoretical basis for improving astrocyte energy metabolism disorders and the treatment of neurodegenerative diseases such as AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a bar chart showing the screening of D-gal modeling dosage and culture time provided in the examples of the present application; compared with the Control group, * indicates P<0.05, and ** indicates P<0.01;

[0010] Figure 2 This is a bar chart showing the effective protective concentration screening of the phytoestrogen components of Erzhi Pills provided in the examples of this application on the D-gal-induced astrocyte energy disorder model; in the figure, Control: cells were not treated; Model: 200mmol / L D-gal was applied for 24h; C1-C4 respectively indicated that the concentrations of Erzhi Pills extracts administered were 0.1, 1, 10, and 100μmol / L. Compared with the Control group, ** indicates P<0.01; compared with the Model group, # indicates P < 0.05,## indicates P < 0.01;

[0011] Figure 3 A bar graph showing the effect of the phytoestrogen components of the Erzhiwan extract provided in the examples of the present application on the glycogen level in the D-gal-induced astrocyte energy damage model;

[0012] Figure 4 This is a bar graph showing the effect of the Erzhiwan extract provided in the examples of the present application on the glycogen level in the D-gal-induced astrocyte energy damage model; compared with the Control group, ** indicates P<0.01; compared with the Model group, ## Indicates P < 0.01; compared with the Lut group, && Indicates P<0.01.

[0013] Figure 5 The glycogen PAS staining results (400×) provided in the examples of the present application, A to F in the figure are: Control group, Model group, Lut group, E2 group, G15 group, LY294002 group;

[0014] Figure 6 The protein expression of GPR30 in each group of sample cells provided in the examples of the present application; A to F in the figure are: Control group, Model group, Lut group, E2 group, G15 group, LY294002 group; compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## Indicates P < 0.01; compared with the Lut group, && Indicates P<0.01.

[0015] Figure 7 The expression of PI3K, Akt, p-GSK3β, and GSK3β proteins in the cells of each group of samples provided in the examples of this application; A to F in the figure are: Control group, Model group, Lut group, E2 group, G15 group, and LY294002 group. Compared with the Control group, ** indicates P < 0.01 compared with the Model group, ## Indicates P < 0.01; compared with the Lut group, && Indicates P<0.01.

[0016] Figure 8 The protein expression of GS in each group of sample cells provided in the present application example; A to F in the figure are: Control group, Model group, Lut group, E2 group, G15 group, LY294002 group. Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ##Indicates P < 0.01; compared with the Lut group, && Indicates P<0.01. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0018] 1 Materials

[0019] 1.1 Cell lines

[0020] C8-D1A mouse astrocytes (Cat. No. ZQ0566) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0021] 1.2 Reagent configuration

[0022] The reagents required for the experiment are prepared as shown in Table 1.

[0023] Table 1 Main reagent configuration

[0024]

[0025]

[0026] 2 Methods

[0027] 2.1 Experimental groups and drug administration

[0028] 2.1.1 In the experiment of screening the effective ingredients and optimal dosage of Erzhi Pills, the groups were divided into the following groups:

[0029] Blank control group: DMEM complete medium, without cells.

[0030] Control group: astrocytes were cultured in DMEM complete medium.

[0031] D-gal injury group: astrocytes were cultured in complete culture medium containing 800 μmol / L, 400 μmol / L, 200 μmol / L, and 100 μmol / L D-gal for 12 h, 24 h, and 48 h, respectively, with 6 replicate wells in each group.

[0032] Drug-treated group (Erzhiwan extract Lut group): astrocytes were co-cultured with 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 100 μmol / L drug solution and the optimal D-gal concentration, with 6 replicate wells in each group.

[0033] 2.1.2 In the study on the mechanism of Erzhiwan extract improving D-gal-induced energy metabolism damage in astrocyte model via GPR30-PI3K-Akt-GSK3β pathway, the groups were as follows:

[0034] Blank group (Control): Astrocytes were not treated.

[0035] Model group: astrocytes were treated with 200 μmol / L D-gal for 24 h.

[0036] Erzhiwan extract group (Lut): Astrocytes were treated with 200 μmol / L D-gal and 10 μmol / L Erzhiwan extract for 24 h.

[0037] Estradiol group (E2): Astrocytes were treated with 200 μmol / L D-gal and 10 μmol / L estradiol for 24 h.

[0038] GPR 30 inhibitor group (G15): Astrocytes were pretreated with 1 μmol / L G15 for 1 h, and then co-treated with 200 μmol / L D-gal and 10 μmol / L Erzhiwan extract for 24 h.

[0039] PI3K-AKT inhibitor group (LY294002): Astrocytes were pretreated with 20 μmol / L LY294002 for 45 min, and then co-treated with 200 μmol / L D-gal and 10 μmol / L Erzhiwan extract for 24 h.

[0040] 2.2 MTT assay to detect cell viability

[0041] The logarithmic phase astrocytes were digested with trypsin and resuspended in DMEM medium at a concentration of 1×10 5 Cells were seeded in 96-well plates at a density of 100 μL per well, and PBS was used to avoid edge effects. After 24 hours of culture, different concentrations of drug solution were added to continue the culture. After treatment, the culture medium was removed, and MTT solution (5 mg / mL, 50 μL / well) was added and incubated for 2 hours. After aspiration, isopropanol (100 μL / well) was added to dissolve the crystals, and the OD value was measured at 570 nm using an enzyme marker to calculate the cell survival rate.

[0042] 2.3 ATP kit screening

[0043] After determining the optimal model and concentration using D-gal and dichlorophen phytoestrogen, 10 6 Up to 10 7Cells were centrifuged at 4000 rpm for 10 minutes at 4°C and the supernatant was removed. The cells were added with 300-500 μL of cold double distilled water, broken in an ice bath, and then in a boiling water bath for 10 minutes. Vortexed for 1 minute for determination. After adding the working solution, the mixture was mixed and allowed to stand for 5 minutes. The absorbance was measured at 636 nm.

[0044] 2.4 Glycogen Kit Screening the Effect of Erzhiwan Phytoestrogen on Astrocyte Glycogen Levels

[0045] Collect 5-10 million cells, centrifuge at 4000rpm for 10 minutes at 4℃, and remove the supernatant. Add 0.75mL of extract solution for ultrasonic disruption, boil in water bath for 20 minutes, cool, add distilled water to 5mL, centrifuge at 12000rpm for 10 minutes at 25℃, and take the supernatant. Add samples and solutions to a 96-well plate, duplicate wells. Add working solution and mix well, and measure absorbance at 620nm.

[0046] 2.5PAS glycogen staining

[0047] After the cells were planted on the slides, they were first washed with PBS, fixed with PAS fixative, stained with periodic acid and Schiff's stain, and finally counterstained with hematoxylin stain. After drying, the slides were sealed with glycerol for microscopic observation.

[0048] 2.6 Kit testing

[0049] Detection of intracellular glycogen, lactate, G6P, ATP levels and HK, GPa activity changes A sufficient number of cell samples were collected according to the kit specifications, and the intracellular glycogen, lactate, G6P, ATP levels and HK, GPa activity of each group were determined according to the kit operation.

[0050] 2.7 Western blot

[0051] The protein levels of intracellular GPR30, PI3K, Akt, GSK3β, p-GSK3β, and GS were detected.

[0052] 2.8 RT-qPCR

[0053] Detect the gene expression of GPR30, PI3K, Akt, GSK3β, and GS in cells.

[0054] 2.9 Data Processing

[0055] All data were expressed as (x±S), and GraphPadPrism 9 software was used for statistical analysis and statistical graph generation. When comparing between groups, if the data conformed to normal distribution, one-way ANOVA test was used, and LSD test was used for pairwise comparison between groups; if the data variance was unequal, Kruskal-Wallis test was used, and P<0.05 indicated significant difference.

[0056] 3 Results

[0057] 3.1 Construction of D-gal-induced astrocyte energy metabolism disorder model and screening of the effective ingredients of Erzhi Pills

[0058] 3.1.1 Screening of treatment conditions for D-gal-induced astrocyte energy metabolism disorders

[0059] In order to determine the optimal conditions for D-gal treatment of astrocytes, the experiment tested the effects of D-gal treatment at different concentrations and times on cell activity and ATP levels. The results showed that ( Figure 1 ), with the increase of D-gal concentration and treatment time, cell activity and ATP level decreased. When treated with 200mmol / L D-gal for 24 hours and 100mmol / L for 48 hours, cell activity and ATP level decreased significantly. Considering the cell survival rate, ATP level and time cost, 200mmol / L D-gal treatment for 24 hours was selected as the optimal condition for causing energy damage.

[0060] 3.1.2 Screening of effective protective concentration of Erzhiwan phytoestrogen on D-gal-induced astrocyte energy disorder model

[0061] In order to find the optimal protective concentration of Erzhiwan extract, the effects of different concentrations of the extract on astrocytes were first tested separately, and then the cells were treated with 200mmol / L D-gal for 24 hours. The results showed that ( Figure 2 ), these components can improve cell viability and ATP levels. Specifically, when the extract concentration was 10μmol / L, co-treatment with D-gal significantly increased cell survival rate and ATP levels. Therefore, this concentration was selected as the optimal condition to protect astrocytes and restore their ATP levels.

[0062] 3.1.3 Effect of Erzhiwan phytoestrogen on glycogen levels in the D-gal-induced astrocyte energy disorder model.

[0063] In order to determine the effect of the phytoestrogen components of Erzhiwan on the glycogen level of D-gal-induced astrocytes, the cells were treated with the optimal concentration determined previously for 24 hours and detected using a glycogen kit. The results showed ( Figure 3 ), compared with the model group, the glycogen level in the drug group was significantly increased (P < 0.01), but there was no significant difference compared with the control group. This shows that the Erzhiwan extract can increase the glycogen content.

[0064] 3.2 Molecular Biology Study of Erzhiwan Extract

[0065] 3.2.1 Glycogen kit and PAS staining results

[0066] Glycogen detection kit and PAS staining results ( Figure 4 , Figure 5 ) showed that compared with the Control group, the glycogen storage in astrocytes of the Model group was significantly reduced (P<0.01); compared with the Model group, the glycogen storage in astrocytes of the Lut group was significantly increased (P<0.01); after administration of the blocker, compared with the Lut group, the glycogen storage in astrocytes of the G15 group and LY294002 group was significantly reduced (P<0.01).

[0067] 3.2.2 Erzhiwan extract has a significant effect on G6P and lactate content in the D-gal-induced astrocyte energy metabolism disorder model

[0068] Compared with the control group, the levels of G6P and lactic acid in the model group were significantly decreased (P<0.01); compared with the model group, the levels of G6P and lactic acid in the Erzhiwan extract (Lut) and estrogen (E2) treatment groups were significantly increased (P<0.01). After using the blocker, the levels of G6P and lactic acid in the G15 and LY294002 treatment groups were significantly decreased compared with the Erzhiwan extract group (P<0.05). The detailed results are shown in Table 2.

[0069] Table 2 Changes in intracellular G6P and lactic acid content in each group of samples

[0070]

[0071]

[0072] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## Indicates P < 0.01; compared with the Lut group, && indicates P < 0.01, & Indicates P<0.05.

[0073] 3.3.3 Effects of Erzhiwan extract on HK, GPa activity and ATP level in D-gal-induced astrocyte energy metabolism disorder model

[0074] The results are shown in Table 3. Compared with the Control group, the HK and GPa activities of the Model group cells were decreased (P<0.01), and the ATP level was significantly decreased (P<0.01); compared with the Model group, the HK and GPa activities of the E2 group and the Lut group were increased (P<0.05), and the ATP level was significantly increased (P<0.01); after the blocker was administered, compared with the Lut group, the HK and GPa activities of the G15 group and the LY294002 group cells were decreased (P<0.05), and the ATP level was significantly decreased (P<0.01).

[0075] Table 3 Changes of HK, GPa activities and ATP levels in each group of samples

[0076]

[0077] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01,

[0078] # indicates P<0.05; compared with the Lut group, && indicates P<0.01, & indicates P<0.05.

[0079] 3.3.4 Erzhiwan extract has a significant effect on GPR30 protein and gene expression in D-gal-induced astrocytes

[0080] The results are shown in Table 4. Compared with the control group, the expression of GPR30 protein and mRNA in the model group was significantly decreased (P<0.01). Treatment with Erzhiwan extract (Lut) and estrogen (E2) significantly increased the expression of GPR30 protein and mRNA (P<0.01). After using the GPR30 blocker G15, the expression of GPR30 protein and mRNA was significantly decreased (P<0.01), while after treatment with the PI3K / Akt blocker LY294002, the expression of GPR30 protein and mRNA did not change significantly ( Figure 6 ).

[0081] Table 4 GPR30 mRNA expression levels in cells of each group of samples

[0082]

[0083] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## Indicates P < 0.01; compared with the Lut group, && Indicates P<0.01.

[0084] 3.3.5 Effects of Erzhiwan extract on D-gal-induced PI3K, Akt, p-GSK3β, GSK3β protein and gene expression levels in astrocytes

[0085] The results are shown in Table 5 and Figure 7 Compared with the control group, the expression of PI3K and Akt in the model group was decreased, while the expression of GSK3β was increased and the expression of p-GSK3β was decreased (P<0.01). Treatment with Erzhiwan extract (Lut) and estrogen (E2) could significantly reverse these changes, increasing the expression of PI3K and Akt, decreasing the expression of GSK3β, and increasing the expression of p-GSK3β (P<0.01). After using GPR30 blocker G15 or PI3K / Akt blocker LY294002, these improvements were reversed, with decreased expression of PI3K and Akt, increased expression of GSK3β, and decreased expression of p-GSK3β (P<0.01).

[0086] Table 5 The mRNA expression levels of PI3K, Akt and GSK3β in cells of each group of samples

[0087]

[0088]

[0089] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## Denotes P < 0.01; && denotes P < 0.01 compared with the Lut group.

[0090] 3.3.6 Effects of Erzhiwan extract on GS protein and gene expression levels in D-gal-induced astrocytes

[0091] The results are shown in Table 6. Figure 8 As shown in the data, compared with the Control group, the expression levels of GS protein and mRNA in the Model group were significantly lower (P<0.01); compared with the Model group, the expression levels of GS protein and mRNA in the Lut group and E2 group were significantly increased (P<0.01); this enhancement effect could be reversed by the GPR30 blocker G15 and the PI3K / Akt blocker LY294002, and the expression levels of GS protein and mRNA were significantly decreased after both treatments (P<0.01).

[0092] Table 6 PI3K, Akt, GSK3β mRNA in cells of each group of samples - Expression level (x±S, n=3)

[0093]

[0094] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## Indicates P < 0.01; compared with the Lut group, && Indicates P<0.01.

[0095] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for improving D-gal-induced astrocyte energy metabolism disorder, characterized in that The method comprises the following steps: a. D-gal-induced astrocyte energy metabolism disorder; b. D-gal-induced astrocytes were treated with dioscorea pellet extract.

2. The method according to claim 1, characterized in that The treatment time is 24 hours.

3. The method according to claim 1 or 2, characterized in that The concentration of D-gal is 200 mmol / L.

4. The method according to any one of claims 1 to 3, characterized in that The concentration of the Erzhiwan extract is 10µmol / L.

5. The method according to any one of claims 1 to 4, characterized in that The method further comprises detecting the intracellular glycogen synthase, glucose-6-phosphate, lactate, ATP levels and the activities of hexokinase and glycogen phosphorylase a.

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