Microspectrophotometer

Total RNA was extracted with TRIzol reagent (e) from PD (n = 3) and EX-PD (n = 3) mouse striatum tissues according to the manufacturer’s protocol. The concentration and purity of RNA were measured using a microspectrophotometer (Tiangen Biotech Co., Ltd., Beijing, China). Quality qualified RNA was frozen at −80°C for subsequent experiments.

TRIzol reagent (Invitrogen Life Technologies, Inc.) was used to extract total RNA from neurons according to the manufacturer’s protocol. The RNA concentration and purity were determined using a microspectrophotometer (Tiangen Biotech Co., Ltd.). RNA was reversely transcribed into first-strand cDNA using the RevertAid First-Strand cDNA synthesis kit (ThermoScientific, Madison, WI, USA). According to the manufacturer’s instruction, the cDNA was amplified using FastStart Universal SYBR Green Master mix on a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Inc.). All primers used in this study were synthesized by Sangon Biotech (Shanghai, China) (Additional file 5: Table S3). GAPDH served as a housekeeping gene, and gene expressions were normalized using the 2^−ΔΔCq method.

The liver tissues of rats were collected from all the groups. The total RNA was extracted using the TRIzol reagents (Invitrogen Life Technologies, Inc.). The microspectrophotometer (Tiangen Biotech Co., Ltd.) was used to detect the concentration and purity of RNA. Briefly, first-strand cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, MA, USA). Then, qRT-PCR analyses of NOX4, NLK, TLR4, IKB, and NF-κB p65 mRNAs were performed with an ABI Q6 real-time PCR machine (Applied Biosystem Inc., MA, USA) using QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) according to the instruction. Every sample was assayed in triplicates. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was served as an internal control. The relative mRNA expression of NOX4, NLK, TLR4, IKB, and NF-κB p65 was determined by the 2-ΔΔCt methods. All primer sequences are displayed in Supplementary Table 1.

TRIzol reagent (Invitrogen Life Technologies, Inc., USA) was used to isolate total RNA from SW480 and NK92-MI cells according to the manufacturer’s instructions. A microspectrophotometer (Tiangen Biotech Co., Ltd., China) was used to determine the concentration and purity of RNA, and qualified RNA was frozen at – 80 °C for subsequent experiments. The RNA was reverse transcribed into first-strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.). Subsequently, qRT-PCR was performed using the FastStart Universal SYBR Green Master Mix on a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. PCR cycling was conducted using the following conditions: 95 °C for 10 min; followed by 45 cycles of 95 °C for 15 s and 60 °C for 60 s; dissociation at 95 °C for 10 s; 60 °C for 60 s; 95 °C for 15 s. All primers used are listed in Additional file 3: Tables S1 and were synthesized by Sangon Biotech (Shanghai, China). GAPDH was used to normalize gene expression, which was measured using the 2^−ΔΔCq method.