Abstract
This study aimed to investigate the effects of curcumin on the expression of antioxidation-related genes in the liver and jejunum and caecum microorganisms of rabbits. A total of 160 IRA rabbits with similar body weight were randomised assigned to 4 groups. The control group received a basal diet, while the experimental groups were given diets supplemented with 50, 100, and 150 g/t curcumin, respectively. The pre-test period spanned 7 days, followed by a 21-day test period. Results revealed a significant upregulation of nuclear factor erythroid-2 related factor 2 (Nrf2) mRNA in the liver and jejunum due to curcumin (p < 0.05). Additionally, curcumin supplementation markedly increased the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNA in the liver and jejunum of rabbits (p < 0.05), and reduced the expression of c-Jun N-terminal kinase (JNK) mRNA in the liver (p < 0.05). The inclusion of 100 and 150 g/t curcumin enhanced caecal microbiota richness and increased the relative abundance of Lactobacillus, Lactobacillus helveticus, and Lactobacillus iners (p < 0.05). Furthermore, curcumin at 100 and 150 g/t significantly increased the relative abundance of coenzyme B biosynthesis and creatinine degradation I (p < 0.05). In conclusion, curcumin’s regulation of antioxidant performance in rabbits appears linked to JNK and Nrf2/NQO1 signalling. Curcumin modulates caecal flora, with probiotic effects mediated by Lactobacillus. Notably, Lactobacillus helveticus and Lactobacillus iners may be beneficial strains associated with curcumin. The recommended curcumin dosage in rabbit feed is 100 g/t.
The regulation of antioxidant performance by curcumin in meat rabbits may be related to JNK and Nrf2/NQO1 signalling.
Curcumin can regulate caecal flora, and its probiotic function may be mediated by Lactobacillus.
Curcumin could improve the coenzyme B biosynthesis and creatinine degradation ability of caecal flora.
HIGHLIGHTS
Introduction
Curcumin, a flavonol compound derived from the roots or stems of the Curcuma species within the Zingiberaceae family (Henrotin et al. Citation2014), has gained increasing recognition in recent years as a valuable functional substance for healthcare and medicine (Amalraj et al. Citation2017). Extensive research has elucidated diverse biological activities associated with curcumin, encompassing anti-oxidative, anti-inflammatory, antiviral, and anti-tumor properties (Coury et al. Citation2021; Islam et al. Citation2021; Momtazi-Borojeni et al. Citation2021; Park et al. Citation2021).
The current intensive farming of rabbits exposes them to several managemental stresses such as early weaning, high stocking density, noxious gases and nutritional imbalance (Liu L et al. Citation2019; Cui et al. Citation2021). Under the effect of these factors, the balance of free radical production and scavenging in the body is disrupted, resulting in excessive accumulation of free radicals, which increases the generation of reactive oxygen species (ROS) (Pisoschi and Pop Citation2015). The accumulation of ROS leads to oxidative stress (OS) in livestock and poultry, which adversely affects intestinal health, immunity, and growth performance (Imbabi et al. Citation2021; Abd El-Hack et al. Citation2022; El-Maddawy et al. Citation2022). Studies have shown that curcumin can protect the organism during oxidative stress and its health effects are attributed to its antioxidant capacity, including scavenging of free radicals, hydrogen peroxide and metal chelation (Ak and Gülçin Citation2008). The body produces a set of endogenous antioxidant defensive systems to prevent ROS accumulation, such as the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant system as well as glutathione (GSH) synthesis and metabolism, which can effectively improve the oxidative stress state of the organism and maintain the stability of the redox state in vivo (Liang et al. Citation2018). In addition, NAD(P)H: quinone oxidoreductase 1 (NQO1), mitogen-activated protein kinase 14 (MAPK14), and c-Jun N-terminal kinase (JNK) have been shown to play key roles in the regulation of oxidative stress (He et al. Citation2022). However, there are fewer reports on the effects of curcumin on antioxidant-related gene expression in meat rabbits.
The intestinal microbiota plays a crucial role in regulating intestinal motility, digestion and absorption, nutrient exchange and immune regulation (Jandhyala et al. Citation2015; El-Saadony et al. Citation2022). In addition, weaned rabbits are very susceptible to microbial invasion, resulting in intestinal microbial disorders, leading to intestinal inflammation, diarrheal and other diseases (Gidenne Citation2015; Placha, Pogány Simonová, et al. Citation2022). The intestinal microbiota can inhibit pathogenic bacterial infections by secreting antimicrobial products, competing for nutrients, maintaining the integrity of the intestinal barrier and regulating phage production (Ducarmon et al. Citation2019). A growing number of studies have shown that curcumin significantly promotes the growth of beneficial intestinal flora, thus realising the biological function of curcumin in regulating intestinal health (Tsuda Citation2018). However, there are no relevant reports of studies on the interactions between curcumin and caecum microecology of meat rabbits.
Hence, the primary objective of this investigation was to assess the expression of antioxidant-related genes within the liver and jejunum of meat rabbits. Simultaneously, alterations in gut microbiota were scrutinised to elucidate the potential molecular mechanisms underlying the impact of curcumin on the organism’s antioxidant capacity and the regulatory role of curcumin in shaping gut microbial composition for its health-related effects. The study specifically aimed to explore the influence of supplemented curcumin on the antioxidant properties of the liver and jejunum, along with its impact on caecum microorganisms in meat rabbits. These findings are intended to furnish valuable insights for the practical application of curcumin in meat rabbit production, offering a novel perspective for investigating its bioactivity.
Materials and methods
Animal welfare statement
All experiments were performed in accordance with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), approved by the Ethics Committee of Animal Experimentation of Hebei Agricultural University (Protocol 2021083).
Animal diets, management, and experimental design
A total of 160 35-day-old healthy meat rabbits with similar body weight were randomly divided into 4 groups with 40 replicates in each group and 1 rabbit in each replicate. The control group was fed a basal diet, and the experimental groups were fed experimental diets supplemented with 50, 100, and 150 g/t curcumin in the basal diet (MSD2, MS1, MS2, and MS3 groups, respectively). The basal diet was formulated according to the feeding standards for rabbits recommended by NRC (1977), and the composition and nutrient levels of the basal diet are shown in Table . The rabbit house and cage were thoroughly cleaned and disinfected before the experiment and were kept in a single cage with three layers of stepped feeding, and each replicate was evenly distributed, each rabbit cage was numbered separately. Food and water were taken AD libitum and fed twice daily at 08:00 and 18:00. The pre-test period lasted for 7 days and the test period lasted for 21 days. All rabbits are injected subcutaneously with inactivated rabbit haemorrhagic disease vaccine at 35 days of age and inactivated Clostridium welchii vaccine at 40 days of age.
Table 1. Composition and nutrient levels of basal diets (air-dry basis).
Sample collection and preparation
On the 21st day of the experimental period, each group of experimental rabbits was weighed on an empty stomach, and 8 experimental rabbits close to the average weight of the group were selected for sampling. After the rabbits were euthanized and slaughtered, in each test rabbit, two portions of caecum were put into a 5 ml cryopreservation tube, and two portions of liver and jejunum tissue were put into a 2 ml cryopreservation tube. All samples were placed in liquid nitrogen, and stored at −80 °C until the final analysis.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Liver and jejunum tissue samples from 5 experimental rabbits were randomly selected for qRT-PCR. Total RNA extraction and qRT-PCR were carried out according to the previously described methods (Cui et al. Citation2021). The RNA was quantified with a biophotometer (Roche, Basel, Switzerland). The PCR data were analysed using the 2-ΔΔCT method (Cui et al. Citation2021). The mRNA levels of the target genes were normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCT) (Wu F et al. Citation2022). The primer sequences are shown in Table . The mRNA sequences were obtained from the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/cgi-bin/genbank).
Table 2. Gene-specific primer sequences used for gene transcription analysis.
Analysis of caecal microflora
The caecum content samples were subjected to 16S rRNA sequencing (Illumina Novaseq Sequencer, 250 PE). Microbial DNA was extracted from the faecal samples using the E.Z.N.A.® DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the manufacturer’s protocols. The V3-V4 region of bacterial 16S rRNA genes was amplified through PCR, utilising the forward primer 338 F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer 806 R (5′-GGACTACHVGGGTWTCTAAT-3′). The components of the PCR reaction contained 5 μl of buffer (5×), 0.25 μl of Fast pfu DNA Polymerase (5 U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 μM) of each Forward and Reverse primer, 1 μl of DNA Template, and 14.75 μl of ddH2O. The thermal cycling process consisted of an initial denaturation step at 98 °C for 5 min, followed by 25 cycles consisting of denaturation at 98 °C for 30 s, annealing at 53 °C for 30 s, and extension at 72 °C for 45 s, with a final extension of 5 min at 72 °C. The resulting PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After quantification, the amplicons were pooled in equal amounts and underwent pair-end 2 × 250 bp sequencing using the Illlumina NovaSeq platform and NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China). The sequencing processes and instruments were provided by Shanghai Personal Biotechnology Co., Ltd. The free online platform of Genes Cloud Platform was utilised for the analysis of the 16S rRNA data (www.genescloud.cn).
Statistical analysis
The gene expression data were analysed and one-way analysis of variance (ANOVA) by Statistical Package for the Social Sciences (SPSS) ver. 26, and differences between means were compared by Duncan’s multiple range test. The results were expressed as Mean ± Standard Error (SE). Statistical differential taxa between four groups were calculated by the Kruskal–Wallis test by SPSS (ver. 26). Significant different levels were presented as p < 0.05.
Results
Effects of dietary supplementation of curcumin on the expression of antioxidant-related genes in the liver of meat rabbits
As shown in Figure , the expression of Nrf2 showed statistically significant differences in the liver of all groups, and the abundance of Nrf2 mRNA was significantly increased in the 50 g/t curcumin (MS1), 100 g/t curcumin (MS2) and 150 g/t curcumin (MS3) groups compared with the control diet (MSD2) group (p < 0.05). Curcumin significantly increased the abundance of NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNA in the liver, with the highest abundance of mRNA in MS2 and MS3 groups (p < 0.05). The difference in abundance of glutathione peroxidase (GSH-Px) and mitogen-activated protein kinase 14 (MAPK14) mRNA in the liver was not statistically significant (p > 0.05). The addition of 50 and 100 g/t curcumin significantly reduced the abundance of c-Jun N-terminal kinase (JNK) mRNA in the liver (p < 0.05).
Figure 1. Effect of curcumin on the expression of antioxidant genes in the liver of meat rabbits (n = 5).
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin; Nrf2, nuclear factor erythroid-2 related factor 2; NQO1, NAD(P)H: quinone oxidoreductase 1; GSH-Px, glutathione peroxidase; MAPK14, mitogen-activated protein kinase 14; JNK, c-Jun N-terminal kinase.
Data are presented as means ± standard error.
a, b Values with different superscripts differ significantly (P < 0.05).
Effects of dietary supplementation of curcumin on the expression of antioxidant-related genes in the jejunum of meat rabbits
As shown in Figure , the expression of jejunal Nrf2 mRNA was significantly higher (p < 0.05) in the curcumin-added MS1, MS2, and MS3 groups compared with the MSD2 group. The expression of jejunal NQO1 mRNA was significantly higher (p < 0.05) in the MS2 and MS3 groups than in the MSD2 group. Consistent with the liver, the addition of curcumin did not significantly affect the expression of GSH-Px and MAPK14 mRNA in the jejunum compared to the MSD2 group (p > 0.05). The expression of jejunal JNK mRNA was not significantly affected in the MS2 and MS3 groups (p > 0.05).
Figure 2. Effect of curcumin on the expression of antioxidant genes in the jejunum of meat rabbits (n = 5).
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin; Nrf2, nuclear factor erythroid-2 related factor 2; NQO1, NAD(P)H: quinone oxidoreductase 1; GSH-Px, glutathione peroxidase; MAPK14, mitogen-activated protein kinase 14; JNK, c-Jun N-terminal kinase.
Data are presented as means ± standard error.
a, b Value with different superscripts differ significantly (P < 0.05).
Effect of dietary supplementation of curcumin on the composition and structure of the caecum microbiota of meat rabbits
The rarefaction curves showed a gentle trend, indicating that the amount of data sequenced in this experiment was appropriate and that the diversity of all samples had been saturated (Figure ). The results of the operational taxonomic units (OTU) rank curve showed that almost all samples were close to saturation, which indicated that the data currently had enough depth to capture the diversity information of the majority of the samples (Figure ). The Venn diagrams reflected the variability between groups, with the MSD2 group-specific OTUs were 16,919, MS1 group-specific OTUs were 17,529, MS2 group-specific OTUs were 15,380, and MS3 group-specific OTUs were 17,231, for a total of 1,284 OTUs across the four groups (Figure ). The α-diversity indices of the gut microbiomes of the groups were compared, and the results showed that the addition of 100 g/t and 150 g/t curcumin significantly increased the Faith_pd index of the caecal flora of broiler rabbits (p < 0.05) (Figure ). The Principal coordinate analysis (PCoA) plots showed that Principal Component Analysis 1 (PCA1) explained 59.7% of the total caecum content bacterial flora community and PCA2 explained 19.7% of all caecum contents flora communities, and the two together explained 79.47% of all flora (Figure ). The above results showed that the addition of curcumin had a significant effect on the microbial colonisation of the caecum contents of meat rabbits.
Figure 3. Curcumin changed the composition and structure of caecal microflora in meat rabbits (n = 8). (A) rarefaction curve. (B) OTU rank abundance curve. (C) Venn of the OTUs in the caecum content among different dietary treatments. (D) Effect of curcumin on α-diversity of caecal microbiota. (E) Weighted UniFrac PCoA plots of the caecal microbiota of different treatments.
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin.
*P < 0.05, **P < 0.01.
The bar graph represents the abundance of species at the gate level for the four groups. As can be seen from the graph, the Top 10 groups in the caecum contents of meat rabbits were Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, Tenericutes, Actinobacteria, TM7, Synergistetes, Cyanobacteria, and Thermi (Figure ). At the genus level, the Top 10 groups of bacteria in the caecum contents of meat rabbits were Oscillospira, Ruminococcus, Akkermansia, Bacteroides, Lachnospiraceae_Clostridium, Coprococcus, Clostridiaceae_Clostridium, Subdoligranulum, Lactobacillus, and Phascolarctobacterium (Figure ). Using the Kruskal-Wallis H-test and pairwise comparisons, it was found that the addition of 100 and 150 g/t curcumin significantly increased the relative abundance of Lactobacillus in the caecal flora of meat rabbits (p < 0.05) (Figure and ).
Figure 4. Curcumin increased the abundance of beneficial bacteria in the caecal microflora of meat rabbits (n = 8). relative abundance of caecal microflora phylum (A) and genus (B) of meat rabbits in different treatments. The relative abundances of dominant caecal microflora (C) phylum and (D) genus of meat rabbits in different treatments.
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin.
*P < 0.05, **P < 0.01, ***P < 0.001.
Potential probiotics associated with curcumin
To further elucidate the effects of different doses of curcumin on the caecal microflora of meat rabbits, we compared the curcumin-added groups with the control group and assessed the differences between the two groups by ‘one-against-all’ further linear discriminant analysis (LDA) effect size (LEfSe) analysis (LDA > 2.5). The results showed that the addition of 50 g/t curcumin increased the relative abundance of o_Rhodobacterales and g_Allobaculum (Figure ). Addition of 100 and 150 g/t curcumin significantly altered the relative abundance of various bacteria, where addition of 100 g/t curcumin significantly increased the relative abundance of o_Lactobacillales, c_Bacilli, g_Lactobacillus, f_Lactobacillaceae, o_Rhizobiales, g_Ochrobactrum, f_Brucellaceae, and f_Streptococcaceae relative abundance (Figure ). The addition of 150 g/t curcumin significantly increased the relative abundance of g_Lactobacillus, o_Lactobacillales, f_Lactobacillaceae, c_Bacilli, g_Allobaculum, c_Erysipelotrichi, f_Erysipelotrichaceae, o_Erysipelotrichales, o_Rhizobiales, f_Peptostreptococcaceae, g_Ochrobactrum, and f_Brucellaceae in relative abundance (Figure ). We found that the addition of both 100 and 150 g/t curcumin increased the relative abundance of o_Lactobacillales, f_Lactobacillaceae and g_Lactobacillus. This further confirms our results that curcumin can regulate Lactobacillus primarily in the gut.
Figure 5. LEfSe analysis of significant difference species in rabbit gut microbiota (n = 8). (A) MS1 vs MSD2. (B) MS2 vs MSD2. (C) MS3 vs MSD2. (left: cladogram diagram; right: LDA diagram; LDA ≥ 2.5).
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin.
Next, we sought to identify potential beneficial bacteria associated with curcumin, and we performed the random forest analysis of the curcumin groups with different doses added separately from the control group to predict the top 20 species that differed between each curcumin dose group and the control group. The results showed that Lactobacillus iners and Lactobacillus helveticus were listed as the marker species for the difference between the MS2 and MS3 groups and the control group (Figure ). The relative abundance of Lactobacillus iners and Lactobacillus helveticus was significantly higher with the addition of 100 and 150 g/t curcumin compared to the control (Figure ). This suggests that Lactobacillus iners and Lactobacillus helveticus may be curcumin-associated beneficial bacteria.
Figure 6. Identification of potentially beneficial bacteria related to curcumin using regression-based random forest algorithm (n = 8). (A) MS1 vs MSD2. (B) MS2 vs MSD2. (C) MS3 vs MSD2. (D) Relative abundance of two potentially beneficial bacteria.
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin.
*P < 0.05, **P < 0.01.
Effect of dietary supplementation of curcumin on the function of the caecum microbiota of meat rabbits
To reveal the effect of curcumin on the functional level of the caecal flora of meat rabbits, we performed PICRUSt2 function prediction and pairwise compared the predicted metabolic pathways among all groups. The results showed that the addition of 50 g/t curcumin did not significantly affect the function of the caecal flora of the rabbit, while the addition of 100 g/t curcumin significantly increased the relative abundance of six metabolic pathways, including creatinine degradation I, coenzyme B biosynthesis, gallate degradation II, methyl gallate degradation, protocatechuate degradation I (meta-cleavage pathway) and L-histidine degradation II. The addition of 150 g/t curcumin significantly increased the relative abundance of three metabolic pathways, including coenzyme B biosynthesis, creatinine degradation I and formaldehyde assimilation I (serine pathway) (p < 0.05) (Figure ).
Figure 7. Curcumin changed the function of caecal microflora in meat rabbits (n = 8). functional pathways predicted by PICRUSt2.
Abbreviations: MSD2, normal diet; MS1, 50g/t curcumin; MS2, 100g/t curcumin; MS3, 150g/t curcumin.
Discussion
Effects of dietary supplementation of curcumin on the expression of antioxidant-related genes in the liver and jejunum of meat rabbits
Several studies have shown that curcumin has good antioxidant activity and is a naturally derived antioxidant (Alizadeh and Malakzadeh Citation2020; Park et al. Citation2021; Nahed et al. 2022; Rafeeq et al. Citation2023). However, there are few studies on the effects of curcumin on antioxidant properties in meat rabbits, and the regulatory mechanism remains unclear. Perker et al. (Citation2019) showed that curcumin can reduce oxidative damage to cells caused by peroxidation by inhibiting hydrogen peroxide, hydroxyl free radicals and lipid peroxidation, thereby improving antioxidant performance. Nrf2 is an important transcription factor for cells to resist oxidative stress and widely exists in various tissues and organs. It can enter the nucleus and interact with antioxidant reaction elements, regulate the expression of a series of detoxification defense and antioxidant genes, and activate a variety of antioxidant enzymes, thereby enhancing the anti-stress ability of cells (Cai et al. Citation2017). In the liver, Nrf2 signalling is one of the core pathways in the antioxidant response of liver cells (Ndisang Citation2017). It has been reported that curcumin can successfully induce the activation and expression of Nrf2 protein in mice, which in turn induces the expression of its downstream target genes and reduces oxidative damage caused by cadmium (Yang et al. Citation2019). NQO1 is a downstream target of Nrf2, and the Nrf2/NQO1 signalling pathway is involved in the process of regulating oxidative stress in the body. Up-regulation of Nrf2 and NQO1 expression can increase the activity of antioxidant enzymes and reduce the level of peroxidation (Yardım et al. Citation2021). Promoting nuclear translocation of Nrf2 can up-regulate NQO1 expression and protect cells from oxidative damage (Wu KC et al. Citation2012). In this study, the mRNA abundance of Nrf2 and NQO1 in the liver and jejunum of rabbits was significantly increased in 100 and 150 g/t curcumin groups. These results indicate that the addition of 100 and 150 g/t curcumin improves the antioxidant capacity of meat rabbits, and Nrf2/NQO1 signalling may be a key target for curcumin to regulate antioxidant capacity. These results suggest that curcumin is involved in the regulation of the antioxidant system in meat rabbits by regulating the expression of Nrf2 and its downstream genes. This is similar to the results reported by Wu et al. (Citation2019) in broiler chickens and by Liu et al. (Citation2017) in rats.
The JNK signalling pathway is a conserved response to intra- and extracellular stress, and ROS can activate JNK signalling in tissue cells (de Los Reyes Corrales et al. Citation2021). Our study showed that the addition of 50 and 100 g/t curcumin significantly reduced JNK expression in the liver of meat rabbits, suggesting that JNK signalling was involved in the process of curcumin regulation of antioxidant properties in meat rabbits. This is similar to the effect of curcumin on rat liver using curcumin by Zhong et al. (Citation2016) and Al-Dossari et al. (Citation2020). Oxidative stress induced by reactive oxygen species (ROS) accumulation can initiate MAPK signalling through the phosphorylation of members of the MAPK family, of which MAPK14 and JNK are the major MAPK subfamilies in mammalian cells (Ki et al. Citation2013). The protein encoded by MAPK14 is a stress-activated protein kinase (also known as p38) (Gupta and Nebreda Citation2015). Our results showed that the addition of curcumin did not affect the expression of MAPK14 in the liver and jejunum of broiler rabbits, suggesting that MAPK14 may not be involved in the regulation of antioxidant properties of liver and jejunum in broiler rabbits by curcumin.
Curcumin alters the structure and composition of the caecum microbiome in meat rabbits
The caecal flora is the basis for the normal functioning of the host’s digestive system, which is large in number, diverse in variety, and interdependent and interactive with the host (Lozupone et al. Citation2012). Gut microorganisms not only directly affect the overall health of the host organism, but also can achieve effects antioxidant by regulating the physiological functions of the host (Blander et al. Citation2017). Calculated Faith’s phylogenetic diversity (Faith_pd) index can reflect the level of population richness (Armstrong et al. Citation2021). In our study, we found that the addition of curcumin at 100 and 150 g/t increased the Faith_pd index of the caecum microbiome, and the PCoA results showed that the curcumin-added group was significantly differentiated from the control group, which is in agreement with the results of a previous study (Shen et al. Citation2017). In a previous study, curcumin elevated microbial richness, and prevented alpha diversity decrease, during colitis and colon cancer prevention (McFadden et al. Citation2015). This has similarities with the results of this experiment, indicating that curcumin can increase the abundance of caecum microbiome and alter the flora structure in meat rabbits.
Lactobacillus may be a curcumin-associated probiotic
Similar to many previous studies, the major phyla in the caecum of meat rabbits were Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, and Tenericutes (Velasco-Galilea et al. Citation2018; Placha, Bacova, et al. Citation2022). At the phylum level, we did not observe curcumin-induced differences in abundance. At the genus level, we found that the addition of 100 and 150 g/t curcumin significantly increased the relative abundance of Lactobacillus in the caecal flora of meat rabbits.
Lactobacillus strains are recognised probiotics (Zhang et al. Citation2018). Lactobacillus have a variety of probiotic functions such as increasing host bioavailability of macro- and micronutrients, improving livestock and poultry growth performance, regulating intestinal microecological balance, and enhancing body immunity (Turpin et al. Citation2010; Shokryazdan et al. Citation2014; Abedin-Do et al. Citation2015). Lactobacillus was capable of activating systemic Nrf2 signalling. Furthermore, oral gavage of the representative Lactobacillus induced Nrf2 in the liver of mice, and this activation was sufficient to protect against acute oxidative liver injury (Saeedi et al. Citation2020). Lactobacillus casei LH23 was able to inhibit the JNK/p-38 signalling pathway and ameliorate DSS-induced colitis (Liu M et al. Citation2020). Among the curcumin-related probiotic strains we further screened, Lactobacillus helveticus was shown to be able to inhibit lymphoma cell proliferation through suppression of the JNK signalling (Hosoya et al. Citation2014). This suggests that curcumin can increase the abundance of Lactobacillus in the caecum and that the effect of curcumin on antioxidant gene expression in meat rabbits may be associated with the increased abundance of Lactobacillus. In addition, it has been shown that the extracellular polysaccharide produced by Lactobacillus helveticus has a strong antioxidant capacity (Li et al. Citation2015; Abid et al. Citation2018). Lactobacillus iners were first described in 1999 by Falsen et al. (Citation1999) in vaginal and urinary tract specimens. It was shown that the unique and thin peptidoglycan layer of the Lactobacillus iners cell membrane may be able to absorb nutrients or secrete proteins more readily than other Lactobacillus species, which can provide essential nutrients to the organism (Kim et al. Citation2020). In addition, Lactobacillus iners has molecular and cellular mechanisms to ferment glucose, maltose, alginate and mannose, of which glucose and maltose are common glycogenolytic products (France et al. Citation2016). Therefore, the addition of 100 and 150 g/t curcumin may have a probiotic effect by enriching Lactobacillus iners in the caecum of meat rabbits and providing more nutrients to the organism. Future studies need to examine the function and classification of active microorganisms as well as culture-based studies to determine the function of these lactic acid bacteria in antioxidant and nutrient provision.
Curcumin alters the function of the caecum microbiome in meat rabbits
Previous reports claimed that coenzyme B is involved in the redox reaction of methanogenic bacteria and releases methane upon reaction with methyl-coenzyme M (Yan and Ferry Citation2018). The excretion of methane not only reduces the volume of intestinal gas but also provides a favourable anaerobic environment for the intestines and promotes the growth of other anaerobic bacteria. Curcumin significantly increased the relative abundance of coenzyme B biosynthesis metabolic pathway in meat rabbits. This suggests that the addition of curcumin may have facilitated methane excretion, thereby promoting the growth of other beneficial bacteria. Creatinine is a product of human muscle metabolism, which is produced in the muscle and then released into the bloodstream and ultimately excreted in the urine, where creatinine accumulates in the body and becomes a toxin that is harmful to the organism (Pundir et al. Citation2019). In our study, the relative abundance of creatinine degradation I pathway was significantly up-regulated by the addition of 100 g/t versus 150 g/t curcumin. A previous study reported that curcumin was able to significantly reduce serum creatinine and thus protect renal function in rats (Xu et al. Citation2021). This suggests that curcumin can enhance the degradation of creatinine by intestinal flora, which is of great significance in the prevention and treatment of kidney injury. Although we have made predictions about the function of gut microbes, future metagenomics sequencing-based technologies to detect functional changes in gut microbes are necessary.
Conclusion
In conclusion, our study reveals that curcumin plays a pivotal role in modulating the antioxidant performance of meat rabbits, primarily through JNK and Nrf2 signalling pathways. Furthermore, curcumin demonstrates a regulatory impact on caecal flora, with its probiotic function likely mediated by Lactobacillus. Notably, Lactobacillus helveticus and Lactobacillus iners emerge as potentially beneficial strains associated with curcumin. Moreover, our findings indicate that curcumin enhances the coenzyme B biosynthesis and creatinine degradation capabilities of caecal flora. Within the confines of our experimental conditions, the optimal curcumin dosage for incorporation into the feed of meat rabbits is determined to be 100 g/t. These insights provide valuable considerations for the strategic utilisation of curcumin in the context of meat rabbit husbandry, offering a nuanced perspective on its functional impact.
Author contributions
BC, MZ and SL designed the study. MZ, SL, FW, ZT, LH, YX, JG and XC collected samples. MZ, SL and FW performed bioinformatic and statistical analysis. MZ, SL and FW wrote the manuscript, BC revised the manuscript. All authors read and approved the final manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Data availability statement
None of the data were deposited in an official repository. The data that support the study findings are available upon request.
Additional information
Funding
References
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