Fecal microbiota transplantation protects rotenone-induced Parkinson's disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis methods
Aim. Evidence-backed execution summary for Fecal microbiota transplantation protects rotenone-induced Parkinson's disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis methods from Fecal microbiota transplantation protects rotenone-induced Parkinson's disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Western blot
reagent used in the protocol.
- Use
- For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APPLYGEN, China) was utilized to lyse the tissues. Then, the lysate was centrifuged at 12,000 g, 4 °C for 20 min to obtain the total prote...
Enzyme-linked immunosorbent assay
reagent used in the protocol.
- Use
- The enzyme-linked immunosorbent assay (ELISA) kits used to detect the levels of mouse TNF-α, IL-1β, IL-6, LPS endotoxin, and lipopolysaccharide binding protein (LBP) were purchased from Jianglai Industrial Limited By Share Ltd, Shanghai, China. The experimental procedures were carried out according to the...
FMT treatment restores BBB and intestinal barrier impairment in the rotenone-challenged mouse model
reagent used in the protocol.
- Use
- Besides, the functions and the structures of the intestinal barriers were also tested. First of all, the in vivo intestinal permeability assay was performed, in which the higher intensity of FD4 detected in the serum represents the elevated gut permeability. The increased concentrations of FD4 were shown in the seru...
Discussion
reagent used in the protocol.
- Use
- Recently, a report further suggested that the increased gut permeability which may cause the motor deficits in the rotenone-intoxicated mice is dependent on the gut microbiota [ ]. Consistently, the results of the bacterial translocation study and the in vivo intestinal permeability assay in our study found that the...
Western blot
For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APPLYGEN, China) was utilized to lyse the tissues. Then, the lysate was centrifuged at 12,000 g, 4 °C for 20 min to obtain the total prote...
- Use
- For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APPLYGEN, China) was utilized to lyse the tissues. Then, the lysate was centrifuged at 12,000 g, 4 °C for 20 min to obtain the total prote...
Bioinformatic analysis
Sequencing data were processed and analyzed by Realbio Genomics Institute (Shanghai, China). Briefly, PANDAseq (V2.9) was used to filter the clean reads by removing the reads with the average Phred score lower than 20, removing the reads containing more than 3 ambiguous bases, and retaining the reads of 250-50...
- Use
- Sequencing data were processed and analyzed by Realbio Genomics Institute (Shanghai, China). Briefly, PANDAseq (V2.9) was used to filter the clean reads by removing the reads with the average Phred score lower than 20, removing the reads containing more than 3 ambiguous bases, and retaining the reads of 250-50...
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Methods
Male C57BL/6J mice aged 8 weeks (20-22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were then acclimatized (12-h light/dark cycle) under standard conditions (temperature 22 ± 2 °C, humidity 50-60%) with ad libitum access to food and water for 7 days. All the procedures were performed following the guidelines developed by the Beijing Municipal Ethics Committee for the care and use of laboratory animals. The animal experiments were approved by the Animal Care & Welfare Committee, Institute of Materia Medica, CAMS & PUMC (No. 00005402).
Methods
The schematic illustration of the animal experimental design is shown as Fig. A. A total of 45 mice were randomly assigned into two groups: the control group ( n = 15) and the model group ( n = 30). In the beginning 4 weeks, the model group received the oral administration of rotenone every day. Meanwhile, the control group mice were administrated with vehicle. After 4 weeks, we randomly divided the model group mice into two groups: Rotenone group ( n = 15) and FMT group ( n = 15). During week 5 and 6, the mice in the FMT group were treated with FMT once per day. In the meantime, the control group and the rotenone group mice received vehicle administration once a day. These mice were daily weighed for 6 weeks. In addition, GI function tests and behavioral tests were performed at week 6. All the mice were sacrificed at week 6 for further analysis. Fig. 1 FMT treatment alleviates motor...
Western blot
For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APPLYGEN, China) was utilized to lyse the tissues. Then, the lysate was centrifuged at 12,000 g, 4 °C for 20 min to obtain the total protein. To separate cytoplasmic and nuclear proteins, the Nuclear/cytoplasmic Isolation Kit (P1201, APPLYGEN, China) was used according to the manufacturer's instruction. As described in our previous study [ ], the protein concentrations were detected by the BCA Protein Assay Kit (P1511, APPLYGEN, China) and western blot was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. The transferred membranes were incubated at 4 °C overnight with the following primary antibodies: mouse anti-TH antibody (1:1000, MAB318, Millipore), rab...
Discussion
However, no therapies targeting the microbiota dysbiosis have been approved by FDA to treat PD patients clinically so far. FMT, a method which introduces gut microbiota from a healthy individual to a patient, can re-establish a stable gut microbial community to treat intractable GI diseases, like inflammatory bowel disease and CDI [ ]. Since accumulating evidence has supported the close interaction between the gut microbiota and the brain [, ], FMT administration has also been applied to some neurodegenerative disorders. For example, a recent investigation demonstrated that FMT administration could protect an Alzheimer's disease (AD) mouse model [ ]. Also, FMT treatment showed neuroprotective effects on a PD patient [ ]. However, the protective effects of FMT administration on PD and the possible mechanisms are scarcely investigated. Based on the significant role of microbiota...
Measurement outputs
What raw and processed outputs should exist?
The schematic illustration of the animal experimental design is shown as Fig. A. A total of 45 mice were randomly assigned into two groups: the control group ( n = 15) and the m...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APP...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
The enzyme-linked immunosorbent assay (ELISA) kits used to detect the levels of mouse TNF-α, IL-1β, IL-6, LPS endotoxin, and lipopolysaccharide binding protein (LBP) w...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Sequencing data were processed and analyzed by Realbio Genomics Institute (Shanghai, China). Briefly, PANDAseq (V2.9) was used to filter the clean reads by removing the reads wi...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
However, no therapies targeting the microbiota dysbiosis have been approved by FDA to treat PD patients clinically so far.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The schematic illustration of the animal experimental design is shown as Fig. A. A total of 45 mice were randomly assigned into two groups: the control group ( n = 15) and the m...; For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APP...; The enzyme-linked immunosorbent assay (ELISA) kits used to detect the levels of mouse TNF-α, IL-1β, IL-6, LPS endotoxin, and lipopolysaccharide binding protein (LBP) w...; Sequencing data were processed and analyzed by Realbio Genomics Institute (Shanghai, China). Briefly, PANDAseq (V2.9) was used to filter the clean reads by removing the reads wi....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
However, no therapies targeting the microbiota dysbiosis have been approved by FDA to treat PD patients clinically so far. FMT, a method which introduces gut microbiota from a h...
from paperReporting output
Report representative outputs alongside summary comparisons for The schematic illustration of the animal experimental design is shown as Fig. A. A total of 45 mice were randomly assigned into two groups: the control group ( n = 15) and the m..., For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APP..., The enzyme-linked immunosorbent assay (ELISA) kits used to detect the levels of mouse TNF-α, IL-1β, IL-6, LPS endotoxin, and lipopolysaccharide binding protein (LBP) w..., Sequencing data were processed and analyzed by Realbio Genomics Institute (Shanghai, China). Briefly, PANDAseq (V2.9) was used to filter the clean reads by removing the reads wi....
inferred from protocolStructured statistical methods
However, no therapies targeting the microbiota dysbiosis have been approved by FDA to treat PD patients clinically so far. FMT, a method which introduces gut microbiota from a h...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
Male C57BL/6J mice aged 8 weeks (20-22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were then acclimatized (12-h light/dark cycle) under standard conditions (temperature 22 ± 2 °C, humidity 50-60%) with ad libitum access to food and water for 7 days. All the procedures were performed following the guidelines developed by the Beijing Municipal Ethics Committee for the care and use of laboratory animals. The animal experiments were approved by the Animal Care & Welfare Committee, Institute of Materia Medica, CAMS & PUMC (No. 00005402).
The schematic illustration of the animal experimental design is shown as Fig. A. A total of 45 mice were randomly assigned into two groups: the control group ( n = 15) and the model group ( n = 30). In the beginning 4 weeks, the model group received the oral administration of rotenone every day. Meanwhile, the control group mice were administrated with vehicle. After 4 weeks, we randomly divided the model group mice into two groups: Rotenone group ( n = 15) and FMT group ( n = 15). During week 5 and 6, the mice in the FMT group were treated with FMT once per day. In the meantime, the control group and the rotenone group mice received vehicle administration once a day. These mice were daily weighed for 6 weeks. In addition, GI function tests and behavioral tests were performed at week 6. All the mice were sacrificed at week 6 for further analysis. Fig. 1 FMT treatment alleviates motor symptoms and gastrointestinal dysfunctions of the rotenone-induced PD mouse model. A The flow chart of animal treatments. B The body weights of mice from week 5 to week 6. C Rota-Rod test. D Adhesive removal test. E Grip strength test. F Pole test. G Intestinal transit distances. H Colon lengths. I...
For total protein extraction, RIPA lysis buffer (C1055, APPLYGEN, China) with protease inhibitor cocktail (P1265, APPLYGEN, China) and phosphatase inhibitors mixture (P1260, APPLYGEN, China) was utilized to lyse the tissues. Then, the lysate was centrifuged at 12,000 g, 4 °C for 20 min to obtain the total protein. To separate cytoplasmic and nuclear proteins, the Nuclear/cytoplasmic Isolation Kit (P1201, APPLYGEN, China) was used according to the manufacturer's instruction. As described in our previous study [ ], the protein concentrations were detected by the BCA Protein Assay Kit (P1511, APPLYGEN, China) and western blot was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. The transferred membranes were incubated at 4 °C overnight with the following primary antibodies: mouse anti-TH antibody (1:1000, MAB318, Millipore), rabbit anti-tumor necrosis factor-α (TNF-α) antibody (1:500, AF7014; Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-interleukin-1β (IL-1β) antibody (1:1000, ab200478; Abcam, Cambridge, MA, USA), rabbit anti-interleukin-6 (IL-6) antibody (1:1000, ab229381; Abcam), rabbit ant...
However, no therapies targeting the microbiota dysbiosis have been approved by FDA to treat PD patients clinically so far. FMT, a method which introduces gut microbiota from a healthy individual to a patient, can re-establish a stable gut microbial community to treat intractable GI diseases, like inflammatory bowel disease and CDI [ ]. Since accumulating evidence has supported the close interaction between the gut microbiota and the brain [, ], FMT administration has also been applied to some neurodegenerative disorders. For example, a recent investigation demonstrated that FMT administration could protect an Alzheimer's disease (AD) mouse model [ ]. Also, FMT treatment showed neuroprotective effects on a PD patient [ ]. However, the protective effects of FMT administration on PD and the possible mechanisms are scarcely investigated. Based on the significant role of microbiota dysbiosis in PD genesis, our study for the first time utilized the chronic oral rotenone-challenged PD mouse model, an appropriate model closely related to gut flora dysbiosis, to evaluate the protective effects of FMT treatment on PD and to elucidate the underlying mechanisms. In our study, FMT tre...
Machine-readable layer
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