Lactobacillus fermentum CQPC08 Attenuates Exercise-Induced Fatigue in Mice Through Its Antioxidant Effects and Effective Intervention of Galactooligosaccharide

Figures & data

Table 1 Sequences of Primers Used in the RT-qPCR Analysis

Gene Name	Sequence
Cu/Zn SOD	Forward: 5ʹ-AACCAGTTGTGTTGTCAGGAC-3’
	Reverse: 5ʹ-CCACCATGTTTCTTAGAGTGAGG-3’
Mn SOD	Forward: 5ʹ-AGACCTGCCTTACGACTATGG-3’
	Reverse: 5ʹ-CTCGGTGGCGTTGAGATTGTT-3’
CAT	Forward: 5ʹ-TGGCACACTTTGACAGAGAGC-3’
	Reverse: 5ʹ-CCTTTGCCTTGGAGTATCTGG-3’
nNOS	Forward: 5ʹ-TCCCAGTAACGGACCTCAG-3’
	Reverse: 5ʹ-TGCTCAACACAGGTTCTATCTCT-3’
iNOS	Forward: 5ʹ-GGAGTGACGGCAAACATGACT-3’
	Reverse: 5ʹ-GCCAAACTTGCTCCATGTCC-3’
TNF-α	Forward: 5ʹ-GAGGCCAAGCCCTGGTATG-3’
	Reverse: 5ʹ-CGGGCCGATTGATCTCAGC-3’
β-Actin	Forward: 5′-CATGTACGTTGCTATCCAGGC-3′
	Reverse: 5′-CTCCTTAATGTCACGCACGAT-3′

Abbreviations: Cu/Zn SOD, cuprozinc-superoxide dismutase; Mn SOD, manganese superoxide dismutase; CAT, catalase; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-α.

Table 2 The Conditions of Each Step of RT-qPCR

Methods	Conditions
System of reverse transcription (Reverse Aid First Strand cDNA Synthesis Kit)	2 μL 0.1 ng - 5 μg total RNA
	1 μL Oligo (dT)18 primer
	9 μL Water, nuclease-free
	4 μL 5× Reaction Buffer
	1 μL RiboLock RNase Inhibitor (20 U/μL)
	2 μL 10 mM dNTP Mix
	1 μL RevertAid M-MuLV RT (200 U/μL)
	Total volume 20 μL
Conditions of reverse transcription	Incubate for 60 min at 42°C → Terminate the reaction by heating at 70°C for 5 min
System of RT-PCR	10 μL SYBR Green PCR Master Mix
	1 μL Upstream primer
	1 μL Downstream primer
	1 μL cDNA template
	7 μL Diethyl pyrocarbonate
	Total volume 20 μL
Cycle conditions of RT-qPCR	Pre-denaturation at 95°C for 3 min → denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s, 40 cycles → dissolution curve was completed at 95°C for 30 s, 60°C for 30 s, and 95°C for 15 s

Table 3 Effects of Lactobacillus fermentum CQPC08 and in Combination with GOS on Body Weight and Liver Indices in Mice with Exercise-Induced Fatigue (N=10/Group)

	Body Weight (g, 0 Day)	Body Weight (g, 35 Day)	Liver Weight (g)	Liver Index (%)
Normal	40.32±1.60^a	44.76±2.98^a	2.04±0.20^a	4.56±0.16^a
Control	39.83±2.23^a	36.74±1.48^e	1.30±0.11^d	3.53±0.21^d
Vc	39.00±1.29^a	40.09±1.01^c	1.70±0.05^b	4.23±0.10^b
CQPC	38.53±1.30^a	40.20±0.47^c	1.69±0.07^b	4.21±0.18^b
CQPC+GOS	39.25±1.72^a	41.82±0.44^b	1.78±0.05^b	4.26±0.07^b
GOS	40.39±1.93^a	38.33±0.17^d	1.53±0.03^c	3.99±0.06^c

Notes: Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).

Table 4 Effects of Lactobacillus fermentum CQPC08 and in Combination with GOS on Exercise Exhaustion Time of Mice

Group	Running Time (Min)
Control	86.00±3.54^e
Vc	128.80±4.38^c
CQPC	139.60±4.34^b
CQPC+GOS	159.40±3.05^a
GOS	92.00±2.12^d

Notes: Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).

Figure 1 Effects of Lactobacillus fermentum CQPC08 and in combination with GOS on the liver morphology of exercise-induced fatigue mice. Magnification 200×. Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle.

Figure 2 The effect of Lactobacillus fermentum CQPC08 and in combination with GOS on the serum levels of BUN, lactic acid, GOT and GPT of exercise-induced fatigue mice. Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).

Figure 3 The effect of Lactobacillus fermentum CQPC08 and in combination with GOS on the serum levels of oxidation index CAT, GSH-Px, T-SOD and MDA of exercise-induced fatigue mice. Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).

Figure 4 The effect of Lactobacillus fermentum CQPC08 and in combination with GOS on the serum levels of inflammatory cytokines iNOS, TNF-α, IL1β and IL-6 of exercise-induced fatigue mice. Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).

Figure 5 The effect of Lactobacillus fermentum CQPC08 and in combination with GOS on the mRNA expression of oxidation- and inflammation-associated genes in the liver. Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).

Figure 6 The effect of Lactobacillus fermentum CQPC08 and in combination with GOS on the mRNA expression of oxidation- and inflammation-associated genes in the skeletal muscle. Normal and control: vehicle (0.9% normal saline); Vc: 200 mg/kg of vitamin C in vehicle; CQPC: Lactobacillus fermentum CQPC08 (1.0 × 10⁹ CFU/mL) in vehicle; CQPC + GOS: CQPC08 (1.0 × 10⁹ CFU/mL) and 200 mg/kg GOS in vehicle; GOS: 200 mg/kg GOS in vehicle. ^a–eDifferent letters indicate that there is a significant difference between the two groups (P<0.05).