Menopause is a natural process that all women will go through, usually accompanied by some physiological and psychological changes, including bone loss, weight gain, depression, nervousness, etc. [1
]. Huge quantities of microfloral communities in the gut play an important role in nutrient absorbance, pathogen defense, immune response, and energy metabolism [4
]. Several studies showed that microfloral communities in the guts of menopausal women or animal models also had significant changes, and some drugs or foods had modulatory effects on these changes [7
Curcumin is an active component of turmeric (Curcuma longa
), used as a spice and a traditional medicine for centuries in Asian countries [10
]. Its anti-inflammatory, antimutagen, antioxidant, and anti-infectious properties have been previously studied [11
]. In a menopausal rat model induced by ovariectomy, curcumin had been widely proven to have weight-loss effects [15
]. However, what effects curcumin exerts on microfloral communities in the intestines of ovariectomized (OVX) rats remain unclear. To answer this question, in the present study, we explored the effect of curcumin on the diversity of gut microbiota in OVX rats for the first time.
2. Materials and Methods
In this study, 18 virgin Wistar rats aged six months and weighing approximately 310 ± 20.0 g were acquired from the Experimental Animal Center in the Academy of Military Medical Sciences (SCXK-(Military) 2016-006, Beijing, China). The protocol involving animals in this study was authorized by The Institutional Ethics Committee of China Academy of Chinese Medical Sciences (Approval number: 2016-017). The sham operation (n
= 6, SHAM group) or bilateral OVX (n
= 12) using a dorsal incision as we reported previously [17
] was performed on the rats after acclimatization. The rats undergoing OVX were divided into two groups based on the treatment agents, including vehicle (OVX group, n
= 6) and curcumin (CUR group, n
= 6). The rats in the CUR group were administered curcumin (Sigma-Aldrich, Saint Louis, MO, USA, dissolved in distilled water) at 100 mg/kg/day by oral gavage. The rats in the SHAM and the OVX groups were administered the same volume of distilled water by oral gavage. All rats were fed standard chow during the course of the experiments (Animal Center of the Fourth Military Medical University, Xi’an, China). The treatment started one week after surgery for 12 weeks. The body weight of each rat was monitored weekly.
2.2. Preparation of Specimens
The day after the last treatment, the animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg body weight) and xylazine (12 mg/kg body weight) and sacrificed by exsanguination. The uterus was dissected out and immediately weighed. Blood samples were obtained by puncturing the abdominal aorta before death and were collected in tubes. Then, serum was prepared by centrifugation (3000 rpm for 10 min) and preserved at −80 °C.
2.3. Measurements of Estradiol in Serum
The estradiol concentration in the serum was detected using an electrochemiluminescence immunoassay (ECLIA) assay kit (Roche Diagnostics, Mannheim, Germany), in accordance with the instructions. All measurements were performed according to the manufacturers’ protocols.
2.4. Fecal Collection and Bacterial DNA Extraction
Rat colons were immediately excised, and fecal samples were harvested for microbial DNA extraction using E.Z.N.A.® Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA), following the manufacturer’s instructions. The quality and quantity of genomic DNA were assessed with a nanodrop spectrophotometer, with the A260/A280 ratio between 1.8 and 2.0 considered a criterion for quality control. No obvious RNA banding was shown by gel electrophoresis, and genomic bands were clear and complete. DNA was frozen at −80 °C prior to PCR amplification.
2.5. PCR Amplification and Sequencing
Fragments encompassing V3–V4 16S rDNA hypervariable regions were PCR amplified from each of the 18 DNA samples using fusion primers (forward: 5′-barcode-GTGCCAGCMGCCGCGG-3′, reverse: 5′-CCGTCAATTCMTTTRAGTTT-3′) and universal primers (forward: 5′-AYTGGGYDTAAAGNG-3′, reverse: 5′-TACNVGGGTATCTAATCC-3′).
The PCR conditions were as follows: 95 °C for 2 min, 25 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, 72 °C for 5 min and holding at 4 °C. PCR products were excisedfrom 1% agarose gels and purified using an AxyPrep DNA GelExtraction Kit (Axygen Biosciences, Union City, CA, USA). The V3–V4 PCR products were sequenced by Illumina MiSeq (Illumina, San Diego, CA, USA).
2.6. Statistical Analysis
Part of quantitative data (weights of bodies and uteri, and level of estradiol in serum) are reported as the mean ± standard deviation; the proportion of gut microbiota is reported as the mean ± standard error. Statistical differences in basal characteristics between the groups were calculated by one-way analysis of variance and q test for continuous variables. p < 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS 19.0 software (IBM Corporation, Somers, NY, USA).
In the current study, compared to SHAM rats, ovariectomy led to significant body weight gain and loss of uterine wet weight in the OVX group rats. However, curcumin had a significant preventive effect on body weight gain rather than loss of uterine wet weight of OVX rats (Figure 1
and Figure 2
). In addition, the results from the ECLIA assay showed that the serum estradiol levels of the SHAM group rats were higher in comparison to the OVX and CUR group rats, which coincided with previous reports [20
]. We inferred that CUR had no estrogen-like action but could be a potential weight loss agent for menopausal women.
Overweight or obesity is a common phenomenon in menopausal women [22
] or rodent models induced by ovariectomy [23
]. The reasons for increasing obesity in menopausal women are still unclear. Some researchers hold that the deficiency of estrogen may be an important obesity-triggering factor [24
]. More and more evidence suggests that the alteration of the microbial community in the intestine may play an important role in the occurrence of obesity [25
], including obesity of menopausal women [26
]. Particularly, Firmicutes
, two major phyla of gut microbiota, are thought to be involved in lipid and bile acid metabolism to maintain systemic energy homeostasis in hosts [27
]. It had been proven that an increase in the ratio of Firmicutes
was correlated with the development of obesity in humans or animal models [30
]. In the present study, compared to SHAM rats, the abundance of Firmicutes
in the gut of the OVX group were significantly increased and decreased, respectively. We speculated that the increased Firmicutes
ratio plays an important role in overweight OVX rats.
At the genus level, compared to OVX group rats, the abundance of Incertae_Sedis
, and Helicobacter
in the gut of the SHAM group rats, and the abundance of Serratia
, and Helicobacter
in the gut of the CUR group rats changed significantly. Some studies suggested that an elevated abundance of Anaerotruncus
in the gut was associated with prenatal stress [33
] and age-related macular degeneration [34
]. Our results showed that curcumin could decrease the abundance of Anaerotruncus
in the gut of OVX rats, and we supposed that the studies exploring the effects of CUR on neuropsychic diseases are prospective.
Very little was known about Helicobacter
in the intestine, and almost all papers were on Helicobacter pylori
. In murine models of gastric cancer or gastritis induced by Helicobacter pylori
infection, ovariectomized animals used to have a more aggravated gastric lesion compared to those without ovariectomy [35
]. Accumulating evidence showed that curcumin was a potent anti-helicobacter pylori
agent in vivo [37
] or in vitro [39
]. In the present study, we found CUR can decrease the abundance of Helicobacter pylori
in the gut of rats. Further research on the relationship between menopause, curcumin, and Helicobacter
in the gut is necessary.