Propolis improves pregnancy outcomes and placental oxidative stress status in streptozotocin-induced diabetic rats

There is an increase in the prevalence of diabetes mellitus (DM) which is projected to increase up to 642 million by 2040. Its effect on the patient socio-economy, physical and medical state has become a major concern globally [1]. Diabetes-induced hyperglycaemic changes have been found to be potentially detrimental to intrauterine foetal development [2]. In an experimental rat, maternal hyperglycaemia-exposed foetus has septal hypertrophy [3]. Moreover, previous study reveals that mothers with pre-gestational DM are more likely to have a baby with some congenital malformations compared with normal healthy women who never have diabetes [4]. Some factors are correlated with foetal anomalies in the diabetic mother which include age, obesity, motivation, educational status, duration of the disease, compliance with diet and medications [5]. Furthermore, oxidative markers such as 8-isoprostane and protein carbonyl levels are significantly higher in placentae obtained from women with gestational DM compared to healthy pregnant women. These data demostrate the presence of oxidative stress in the placentae of women with hyperglycaemia which may potentially lead to foetal compromise [6]. Antioxidants prevent or remove oxidative damage to a target molecule through donating an electron to stabilise the free radical [7]. Consumption of natural products with antioxidants such as flavonoid and phenolic acids improves oxidant-antioxidant status and decreases the risk of developing DM complications [8]. Superoxide anion radical (O⁻ ₂) is one of the strongest reactive oxygen species (ROS) among the radicals that are generated after oxygen is taken into living cells. This O⁻ ₂ can change to other harmful ROS, which lead to oxidative stress and implicated in the aetiology of many diseases including DM [9].

Brazilian propolis at doses of 100, 200 and 300 mg/kg body weight for 40 days improve body and kidney weights, serum glucose, lipid profile, malondialdehyde (MDA) and renal function in diabetic male rats [10]. Saudi Arabian propolis at 200 mg/kg/day reduces fasting blood glucose (FBG) and MDA levels, and preserves pancreatic islet of Langerhans in diabetic rats and alleviates diabetic signs [11]. Turkey propolis at doses of 0.2, 0.4 and 0.6 mg/ml per day for 28 days significantly improve cryopreservation and fertilization ability in fish semen [12]. Malaysian propolis at 300 mg/kg/day for 4 weeks has shown antihyperglycaemic property in diabetic rats [13]. However, to date, it is known whether propolis may also ameliorate hyperglycaemia-induced impaired pregnancy outcomes and placental oxidative stress in DM. Hence, the objective of the study was to determine the effects of propolis alone or combined with insulin on maternal status, pregnancy outcomes and placental oxidative stress status in streptozotocin-induced diabetic female rats.

In this study, we investigated the effects of propolis alone as monotherapy or in combination with insulin on maternal status, pregnancy outcomes and placental oxidative stress status in diabetic female rats. Considering that DM is a disorder of multiple etiologies, monotherapy with an antihyperglycaemic agent may not be effective [14]. Initial FBG levels of all the STZ-induced diabetic rats were greater than 200 mg/dL and significantly higher than non-DM group prior to the commencement of treatments showing the establishment of diabetic animal model. Final FBG in DM + Propolis group was comparable with DM + insulin group which may indicate that propolis and insulin are equally effective to produce a comparable antihyperglycaemic effect. The antihyperglycaemic effect of 300 mg/kg/day Malaysian propolis for 4 weeks found in the present study is in line with the findings on propolis from other countries. For instance, Nigerian propolis at doses of 200 and 300 mg/kg/day for 4 weeks [15] and Saudi Arabian propolis at a dose of 300 mg/kg/day for 2 weeks significantly reduce FBG level in DM male rats [11]. Moreover, Egyptian and Brazilian propolis at the dose of 300 mg/kg/day [16] and 900 mg/kg/day [17], respectively, for 2 weeks significantly reduce FBG level in DM male rats, which further support propolis as a potential antihyperglycaemic agent possibly due to the ability of polyphenols in propolis to stimulate insulin secretion by the remaining islets cells with normal function [11]. Furthermore, the antihyperglycaemic effect was found to be more pronounced in DM + Combined group compared with DM + propolis and DM + Insulin groups suggesting that propolis in combination with insulin may produce greater antihyperglycaemic effect than propolis or insulin alone as mono-therapy.

DM may alter reproductive functions in human subject [18]. The longer conception period in DM group compared to non-DM group may be due to the presence of hyperglycaemia which is associated with reduced libido as previously shown in DM male rats [19]. However, the conception period was significantly lower in DM + Combined group, not in DM + Propolis and DM + Insulin groups, compared with DM group. These findings may indicate that propolis combined with insulin is able to improve conception period which could be attributed to the concomitant greater improved FBG level found in DM + Combined group. The significant improved maternal body weight gain in DM + propolis, DM + Insulin and DM + Combined groups could also be attributed to the antihyperglycaemic effect of propolis and insulin. The improved maternal body weight in DM + propolis is in accordance with previous study [13] and propolis has been shown to improve insulin level in diabetic rats [11]. This in turn may improve maternal body weight as insulin is known to exert an anabolic effect through stimulating protein synthesis and inhibiting protein degradation and lipolysis [20].

The significantly higher implantation losses might explain for the lower number of foetus found in DM group compared to non-DM group. These findings support the fact that chronic exposure to hyperglycaemia during early pregnancy is associated with implantation loss [21]. Nevertheless, failure of embryo implantation may cause morphological alterations of embryo that eventually interferes with the implantation and promotes embryo lethality. Additionally, failure of implantation is also resulted from disturbance in tubal transport and subsequent interference in the time of blastocyst arrival in the uterus [22]. These findings support the fact that DM in the first trimester of pregnancy is detrimental to blastocyst and embryo formation [23]. Pre-implantation loss was significantly lower in DM + insulin and DM + Combined group compared to DM group which may suggest that propolis in combination with insulin has greater effect in reducing pre-implantation loss as compared to propolis alone. However, the significantly improved post-implantation loss and number of foetus might suggest the comparable action of propolis, insulin or in combination possibly via their antihyperglycaemic effect to prevent post-implantation loss in this diabetic animal model. These findings are similar with a study using an essential acid taurine that significantly improves pregnancy outcomes and protects both the dam and embryos during pregnancy in diabetic rat [23]. The findings on normal gross morphology of the foetuses are in contrast with a few studies in which prenatal stress in rodents is associated with the risk of developing congenital abnormalities [24]. The external evaluation in the present study might suggest that propolis does not cause any foetal gross malformation and assessment on internal organs is, however, suggested in further study.

Foetal blood glucose level was significantly higher in DM group and this could be associated with STZ-induced maternal hyperglycaemia and hypoinsulinaemia despite normal beta cells function in the foetus. However, foetal blood glucose was significantly improved when treated with propolis or insulin without significant change in foetal body weight. The reduction in foetal birth weight in diabetes may be associated with the detrimental effect of maternal hyperglycaemia leading to intrauterine growth retardation [25, 26]. However, both foetal blood glucose and foetal body weight were significantly improved in DM + Combined group which could be attributed to the improved maternal FBG and weight.

Placental MDA and PCO levels were significantly lower while placental TAC level was significantly higher in DM + propolis, DM + Insulin and DM + Combined groups compared to DM group which may suggest the ability of propolis and insulin to reduce placental oxidative stress. This finding may support previous study showing the antioxidant potential of Iranian propolis in DM rats [27] and the greater beneficial effects in DM + Combined group may suggest the synergistic effect of propolis and insulin. The reduced placental oxidative stress could be indirectly due to the antihyperglycaemic action of propolis and insulin as there is a presence of placental oxidative stress in mother with hyperglycaemia [6]. Apart from that, the antioxidant effect of propolis found in the present study could also partly due to the direct synergistic actions of flavonoids and phenols, which have antioxidant property, that present in Malaysian propolis [28].

In conclusion, this study may suggest the protective effects of propolis against DM-induced impaired pregnancy outcomes and placental oxidative stress with greater effects when combined with insulin which could be partly due to the synergistic effect of some of its phytochemical constituents. Apart from supporting the traditional belief on the beneficial effect of propolis on health, further studies are suggested to determine its molecular mechanism of action and its possible role in protecting or reducing complications of DM in other organs.