Protective Effect of Caffeic Acid on Streptozotocin Induced Gestational Diabetes Mellitus in Rats: Possible Mechanism

Clinical studies suggest that the diabetes affects the >5% of pregnant women during the pregnancy, inducing the abnormalities in the reproductive that enhance the risk of congenital anomalies, intrauterine growth retardation, spontaneous abortion, mortality and morbidity of both newborn/fetus and mother (Wang et al., 2019). Gestational diabetes mellitus (GDM) is a typical condition during the pregnancy due to insufficient production of insulin. GDM seriously spoils the maternal and infant health (Alfadhli, 2015). Previous studies suggest that the patient suffering from the GDM usually did not show any sign and symptom of diabetes erstwhile to pregnancy, and frequently detected during the 2nd trimester of pregnancy, with 3-5 % of women even suffering long term diabetes mellitus after the pregnancy (Coustan, 2013; Lefkovits et al., 2019). During the expansion of GDM during the maternal period, showed the effect on the fetal and exhibited the development of abnormal fetal. Even though hyperglycaemia is major factor, take part in the pathogenesis of diabetic embryopathy, and increase the incidence of malformations (Bain et al., 2015; Maresh, 2005). It is well proofed that the incidence of fetopathy and embryopathy increased due to oxidative stress and during the diabetes and other metabolic disturbance induced the generation of oxidative stress (Maritim et al., 2003; Spaight et al., 2016). The most protuberant anti-teratogenic effect was accomplished via anti-oxidant therapy, it’s not decrease the incidence the teratogenic, also control the glycaemic control, this is beneficial for both outset and during pregnancy (Hong et al., 2017; Yao et al., 2015). Various published research suggests that the supplementation with transgenic of Cu/Zn superoxide dismutase or antioxidants such as vitamin C and vitamin E could be valuable in improvement of diabetes linked teratogenic effect and oxidative stress. Several researchers make attempt to use the natural phyto-constituent in mitigation of oxidative stress and thus decrease the embryopathy under the diabetic conditions (Hong et al., 2017; Yao et al., 2015; Zou et al., 2018).

Caffeic acid (CA), 3,4-dihydroxycinnamic acida natural phenolic phyto-constituent widely present in the several medicinal plants such as vegetable, fruits, olive oil, wine and also present in the human plasma in various concentration depend upon the diet (Peyrat-Maillard et al., 2003; Sato et al., 2011). Its synthetic derivative also available and posses multiple activity including anticancer, anti-inflammatory, antifibrosis, antihypertensive, metal chelating agent, antioxidant, antiviral, antidepressive, antidiabetic and antibacterial (de Souza and Spinelli, 2009; Sul et al., 2009). CA also reduces the multiple inflammatory pathways against the various cell lines due to its anti-inflammatory nature. It also suppress the LPS induced inflammation against the Raw 264.7 cells via inhibition of p38 mitogen-activated protein kinases (p38MAPK), NH-2 terminal kinase ½ activation, nuclear factor Kappa B (NF-kB) (Chen and Ho, 1997; Rajendra Prasad et al., 2011). However, its fetal development using rat model of gestational diabetes mellitus remain unclear. Hence, the aim of the current experimental investigation to scrutinize the fetal protective effect of caffeic acid on GDM induced rats and explores the possible mechanism of action.

Streptozotocin and caffeic acid was purchased from the Sigma Chemical Co. U.S.A. Comassie blue, sodium citrate, etheylenediamine tetra acetic acid (EDTA), glutation, 5,5 dithiobis-2-nitrobenzoic acid (DTNB), tris-Hcl, morpholinopropansulfonic acid (MOPS), 4,2 hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), ethyleneglycol- bis (2-aminoethylether) -N,N,Ń,Ń- tetraacetic acid (EGTA), magnesium chloride (MgCl2), hydrochloride (HCL), potassium chloride (KCl), sodium hydroxide (NaoH), n-butanol, 2´7´ - dicholorofluoresceindiacetate (DCFH-DA), ethylacetate, thiobarbutiric acid (TBA), trichloroacetic acid (TCA), phosphoric acid, 2,4 dinitrophenylhydrazine (DNPH) and guanine hydrochloride.

Female and male Wistar rat weight 150-180 g 10-12 old used for the current investigation study. The Wistar rats were received from the animal house and kept in the standard condition (20±5°C temperature; 60-80% relative humidity; 12 h light and 12 h dark cycle). The rats were received the standard pellet chow and water ab libitum. All the experimental protocol performed according to the protocol approved by the University animal ethical committee.

Briefly, the weight of the experimental rats was maintained between 240-280 g at 12 weeks. The rats having the blood glucose level (BGL) more than the 6.8 mmol/L excluded from the current experimental study. Both male and female rats were allowed to mate and female deficient pessary next day was considered pregnant for 0.5 days. Intravenously (tail vein) administration of streptozotocin (STZ) 25 mg/kg (dissolve in the citrate buffer, pH=4.5) was used for induce GDM and untreated pregnant female rats were used as normal control in the current experimental study (Wang et al., 2019).

After induction of STZ administration, the blood was collected from the retro orbital and blood glucose was estimated at regular time interval via using the glucose oxidase method with minor modification. After successfully induction the diabetes, the female rats were anesthetized and embryos were excised on the ice and some embryos was homogenized in the phosphate buffer saline (PBS) and centrifuged at 8000 rpm at 4°C for 10 min and stored in the deep refrigerator at -20°C.

For the estimation of fetal development, the plasma insulin, blood glucose level, plasma insulin, RAGE and AGEs level in the different tissue of the pregnant rats were determine at regular time interval via using ELISA kits following the manufacture instruction. At end of the experimental protocol, the placenta and foetuses were successfully separated and the weight and number of placenta and foetuses were recorded. Finally, the microscopical evaluation was performed to determine the malformations and defects of each organ of fetal (Tang et al., 2014; Wang et al., 2019).

Glycosylated haemoglobin A1c (HbA1c), serum C-peptide (CP), hexokinase, hepatic glycogen, glucose-6-phosphatase, fructose-1,6 biphosphatase (Kumar et al., 2013a, b) and free fatty acid (FFA) were estimated using ELISA kit following the manufacturer’s instruction.

Lipid parameters such as triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL) and very low-density lipoprotein (VLDL) were estimated using colorimetric kits following the manufacturer’s instruction (Abcam).

Antioxidant parameters such as superoxide mutase (SOD), glutathione (GSH), catalase (CAT), protein carbonyl (PC), malonaldehyde (MDA) and glutathione peroxidase (GPx) were estimated via using the previous method with minor modification in the hepatic and pancreas tissue (Shivananjappa and Muralidhara, 2012).

One-way ANOVA was used for the multiple comparisons. Graph pad prism was used for statistical analysis. P<0.05 was considered as statistically significant.

Figure 1A demonstrated the effect of caffeic acid on STZ induced GDM rats. Normal control group rats showed the almost similar trend in the blood glucose pattern till end of the experimental study. STZ induced GDM group rats showed the increased blood glucose level and increased blood glucose level remain increased till end of the experimental study. STZ induced GDM group rats treated with the caffeic acid significantly (P<0.001) down-regulated as compared to the STZ induced GDM rats.

Body weight of all group rats presented in Figure 1B. Figure 1B exhibited body weight of rats before and after the pregnancy. Normal group pregnant rats demonstrated the increased body weight before and after the pregnancy. STZ induced GDM group rats showed the decreased body weight as compared to normal control rats. Dose dependently treatment of caffeic acid significantly (P<0.001) increased the body weight as compared to STZ induced GDM rats.

The AGEs level of normal control group rats remains unchanged till complete experimental protocol. On the country, STZ induced GDM rats exhibited the increased level of AGEs as comparison to normal control group rats. STZ induced GDM rats showed reduced level of AGEs at dose dependent manner (Fig. 1C).

Figure 2 exhibited the effect of caffeic acid on HbA1c, serum C-peptide, free fatty acid, FINS and hepatic glycogen. Normal control group rats showed the almost normal level of biochemical parameters. STZ induced GDM rats showed the increased level of HbA1c, serum C-peptide, free fatty acid, FINS and dose dependent manner of caffeic acid showed the reduction the level of HbA1c, serum C-peptide, free fatty acid and FINS (Fig. 2).

An opposite trend was observed in the level of hepatic glycogen in the STZ induced GDM rats and dose dependently treatment of caffeic acid significantly (P<0.001) enhanced the level of hepatic glycogen.

Figure 3 illustrated the effect of caffeic acid on the fetuses weight, placental weight and placental index. STZ induced GDM group rats showed the decreased fetuses wight and increased the level of placental weight and placental index. Caffeic acid significantly (P<0.001) increased the fetuses weight and reduced the placental weight and placental index at dose dependently manner

Normal control group rats showed the normal pattern of lipid parameters till end of the experimental study. STZ induced GDM rat showed the increased level of TC, TG, LDL, VLDL and decreased level of HDL and concentration dependent treatment of caffeic acid significantly (P<0.001) altered the level of lipid parameters (Fig. 4).

Figure 5 showed the effect of caffeic acid of the antioxidant parameters (liver and pancreas) on normal control and STZ induced GDM rats. Normal control group rats showed the almost similar level of antioxidant parameter. STZ induced group rats showed the increased level of MDA and reduced level of SOD, CAT, GPx, GSH in the liver and pancreas as compared to normal control. STZ induced GDM rats received caffeic acid significantly (P<0.001) reduced the level of MDA and increased the level of SOD, CAT, GPx, GSH in the liver and pancreas.

Clinical studies suggest that the GDM affects the 10% of pregnant women and women suffer from the GDM having the higher chance to develop the long-term type 2 diabetes mellitus. The prevalence of GDM is enhancing globally due to recent trend in advance maternal stage and obesity (Shivananjappa and Muralidhara, 2012). Women suffering from the GDM are the high risk to develop the infection and pregnancy complications including ketoacidosis and hypertension. GDM suffer pregnant women on the risk for advance pregnancy complications such as spontaneous abortion, fetal abnormalities, macrosomia, still birth and intrauterine growth restriction (Vafaei-Pour et al., 2018). The frequency of congenital malformations of fetal vital organs is as high as compared to the without complicated pregnancy (Vafaei-Pour et al., 2018). Lot of research conducted on the diabetes, but few of the researcher focus their research only the GDM. However, in the current experimental protocol, we made attempt to estimate the protective effect of caffeic acid on GDM induced via streptozotocin and explore the possible mechanism.

Various researches suggest that the oxidative stress play an important role to develop the cognitive anomalies (Lappas, 2014; Wang et al., 2019). There is enhancing the evidence that showed ROS have a significant role in the pathology of various congenital anomalies such as produced via GDM, cocaine, alcohol intake and radiation (Szmuilowicz et al., 2019). Various teratogenic drugs viz., thalidomide and phenytoin showed the similar mechanism of action. Currently, much work showed that the oxidative stress plays a significant role the diabetes and pathogenesis of diabetic complications (Shivananjappa and Muralidhara, 2012). Clinical and experimental researches have shown that hyperglycemia through various biochemical pathways such as protein glycation and autoxidation leads to boost the formation of ROS (Vafaei-Pour et al., 2018). Similarly, it has been appeared that developing the embryos are very sensitive to high concentration of ROS, particularly during the organogenesis stage.

During the GDM, increased the level of ROS in the mothers due to increase level of protein oxidation, lipid peroxidation (LPO), ribonucleic acid (RNA) and deoxy ribose nucleic acid (DNA) damage in the expansion of embryo (Hong et al., 2017; Yao et al., 2015; Zou et al., 2018). These pathological conditions lead to the more prevalence of skeletal, cardiovascular, brain and other abnormalities in the offspring of diabetic women as compared to the non-diabetic women. In the current experimental study, we observed that the enhanced level of ROS, protein carbonyl and lipid peroxidation and destruction of endogenous enzymes (enzymatic and non enzymatic) antioxidant in the embryo of diabetic rats. Simultaneously, there was a correlation was observed between the degree of oxidative stress and rate of anomalies in embryo (Hong et al., 2017; Yao et al., 2015). Moreover, it has been shown that the controlling the glycaemic control, the rate of diabetic complications remain still high. Still the researcher targeting the diabetes and diabetic complications, but not targeting the oxidative stress induced diabetic complications (Wang et al., 2019). In this current experimental study, we targeting the oxidative stress and its induced the diabetic complication to treat the GDM. In the current experimental study, treatment group reduced the GDM at dose dependent manner due to its antioxidant manner. In the current experimental studies, we observed that the significantly boost the reactive oxygen species (ROS) production in the embryo, protein oxidative and LPO and increased the embryonic antioxidant capacity in the diabetic rats. It is well documented that during the hyperglycemia, increase the generation of free radical in the body and various researches suggest that the degradation of glycated proteins, glucose oxidation and non-enzymatic glycation are responsible for the production of free radicals. During the production of free radicals, endogenous antioxidant such as CAT, SOD, GPx and some vitamins play a significant role to scavenge the free radicals and prevents the side and toxic effects (Lappas, 2014; Wang et al., 2019). Previous investigation suggests that the expansion of embryos is very sensitive to enhance the ROS level, especially during the organogenesis stage. CAT and SOD are the first line endogenous antioxidant enzymes and take part important role in the reduction of ROS in the body (Hong et al., 2017; Yao et al., 2015; Zou et al., 2018). The first line endogenous antioxidant scavenges the free radical, ½ O2, and OH and reduced the oxidative stress. During the GDM induced a similar result was obtained and dose dependent treatment of caffeic acid reduced the free radical formation via increased the level of endogenous antioxidant. Studies suggest that the ROS is the 2nd messenger and its take part to generate the various redox sensitive signaling pathways (Hong et al., 2017; Zou et al., 2018). The most importantly, its induces the phosphorylation of insulin receptor and substrate in the insulin signalling pathways, subsequently leading to reduced the activity of signal molecules and deteriorate the insulin sensitizing effect and induce the insulin resistance. In the current protocol, we observed that the caffeic acid considerably removed the ROS effect via 2 mechanisms, it could be increase the level of endogenous antioxidant and boost the insulin sensitizing effect and decrease the insulin resistance (Hong et al., 2017; Zou et al., 2018). This effect may be generated due to decrease the blood glucose level in the experimental rats and increase the level of insulin.

In summary, the caffeic acid is a powerful antioxidative substance, during the GDM, the excessive generation of free radical and oxidative stress and due to the nature of antioxidative, caffeic acid considerably inhibited the free radical production and oxidative stress. In future, we scrutinized the possible mechanism of action.

The authors declare no conflict of interest.