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
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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...
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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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