Sterculia tragacantha Lindl Aqueous Leaf Extract Ameliorate Cardiomyopathy in Streptozotocin-induced Diabetic Rats via Urotensin II and FABP3 Expressions.

Sterculia tragacantha (ST) Lindl leaf is commonly used locally in the management of diabetes mellitus (DM) and its complications. This study was aimed at assessing the valuable effects of ST leaf on streptozotocin-diabetic cardiomyopathy (DCM). Streptozotocin was administered intraperitoneally to the experimental animals to induce DM, and hence, placed on different doses of ST for 14 days. Thereafter, on the 15th day of the experiment, the animals were euthanized, and a number of cardiomyopathy indices were investigated. The diabetic rats exhibited a momentous increase in hyperlipidemia, lipid peroxidation as well as a significant (p < 0.05) decline in antioxidant enzyme activities. The serum creatine kinase MB (CK-MB), C-reactive protein (CRP), cardiac troponin I, tumour necrosis factor-alpha (TNF-α) and urotensin II expression revealed a significant (p < 0.05) upsurge in diabetic rats. Also, the expression of GLUT4 and fatty acid-binding protein 3 (FABP3) were significantly (p < 0.05) reduced in diabetic rats. However, at the conclusion of the experimental trial ST significantly (p < 0.05) attenuated hyperlipidemia, oxidative stress biomarkers by augmenting the antioxidant enzyme activities and decrease in lipid peroxidation, ameliorated CK-MB, CRP, cardiac troponin I, TNF-α, and urotensin-II levels, and improved GLUT4 and FABP3 expressions. Similarly, the administration of ST prevented histological alterations in the heart of diabetic animals. Therefore, the obtained results suggest that ST could mitigate DCM in streptozotocin-induced diabetic rats.

II with downregulation of fatty acid-binding protein 3 FABP3 which negatively regulates the cell cycle and inhibits proliferation of myocardial cells leading to heart disease e.g. cardiomyopathy 6,7 . Urotensin II is a vasoconstrictor and even more potent than endothelin-1 and is one of the several neurohormonal systems activated in heartrelated diseases. FABP3 is known as a 14 to 15 kDa protein mainly expressed in the heart and some other organs 8 . FABP3 has the ability to binds long-chain fatty acids and then transports them to specific cell organelles for lipid storage and metabolism as well as signaling transduction and transcriptional regulation 7 . These abnormalities encourage inflammation biomarkers e.g. C-reactive protein as well as the activities of other biomarkers that are paramount to the heart tissue such as Creatin kinase MB, serum lipid profiles, etc. .
In order to mimic what happens in human beings, DM was induced by introducing a single dose of streptozotocin diabetogenic chemical into the experimental animals. This triggers selective destruction of pancreatic beta cells which are insulin-producing cells 1 .
Currently, there are no perfect medications that can cure DM, and they are also associated with series of drawbacks such as fatty liver, anorexia, insulin resistance amongst others 9 . However, medicinal plants have been endowed with different phytochemicals which are useful as a remedy for human diseases. Locally, various herbs have been endorsed in the management of diabetes mellitus and its complications especially DCM. In this regard, an example is Sterculia tragacantha; the family of Malvaceae called Uhobo and Abalo among Eastern and Western people of Nigeria. Sterculia tragacantha has been documented in the treatment of boils, diarrhea, dyspepsia, gout, fever, gonorrhea, snake bite, syphilis, tapeworm, gout, whitlow, etc. especially in rural communities 10,11 . Added to this, without proper scientific barking, the leaf of this plant is used locally in some communities in the Western and Eastern parts of Nigeria in the management of diabetes mellitus and its complications. Hence, there is little/no information on the effectiveness of this plant in the treatment/management of diabetic cardiomyopathy which is the focus of this study.

Plant and its preparation
The leaves of Sterculia tragacantha were acquired from a local farm in Osogbo, Osun State, Nigeria, and scientifically defined at Forestry Research Institute of Nigeria FRIN , Ibadan, Oyo State, Nigeria with voucher number FHI:112856. Hence, the leaves were shade dried for three weeks before being pulverized into a powder form via an electric blender. Subsequently, five hundred grams of the powder sample was extracted in 5 liters of distilled H 2 O 2 and occasionally stirred at room temperature, sieved via Whatman No. 1 filter paper. Then a freeze-dryer was used to concentrate the obtained filtrate and the acquired yield was weighed and named ST Sterculia tragacantha . This was scrapped into a sample bottle and used for various analyses.

Chemicals/Reagents
Sigma-Aldrich, Inc. St Louis MO provided the chemicals and reagents employed in this study St Louis MO . In addition, the primers were procured from Inqaba Biotech South Africa . Furthermore, enzyme kits were acquired from Randox USA .

Experimental animals and induction of diabetes mel-
litus A known number 50 of male albino rats of six to eight weeks old were acquired from Ikere-Ekiti Suramos farm , Ekiti State, Nigeria of 130-146 g in weight. The animals were acclimatized for a week and permitted to food and drinking water. The protocol of Afe Babalola University Animal Ethical Committee ABUAD/2020/SCI/BCH/011 was strictly abided throughout the experimental duration.
The animals fasting blood glucose levels were studied after overnight fasting. Following that, the rats were injected intraperitoneally via a single dose 35 mg/kg of freshly prepared streptozotocin STZ using 0.1 M citrate buffer at pH 4.5 . At exactly 48 hours after induction, each rat fasting blood glucose level was measured by employing a Fine test TM glucometer. Surprisingly, all the induced animals had ≥200 mg/dL fasting blood glucose levels and are diabetic as previously reported by Ajiboye et al. 12 .

Animal grouping
Five groups of animals ten animals in each group based on the body weight were grouped as follows: Group A: Non-diabetic rats given distilled water normal control Group B: Diabetic rats without treatment diabetic control Group C: Diabetic rats given a low dose 150 mg/kg body weight of ST LDST DR LDST Group D: Diabetic rats given a high dose 300 mg/kg body weight of ST HDST DR HDST Group E: Diabetic rats given 200 mg/kg body weight of metformin ME DR MET Note that the ST and MET were given to the respective animals via syringe and canular with a round ball-like bead at the tip . Also, the doses employed was established from the earlier report by Ajiboye et al. 13 .

Sacri ce, collection of blood and hearts
At the end of the intervention period of two weeks, the rats were sacrificed by cervical dislocation as previously documented by Ajiboye et al. 13 . Blood was collected from rats into plain bottles and centrifuged at 1500 rpm for 30 minutes. Then the hearts were harvested and washed using 0.9 NaCl. The remaining parts of the heart were homogenized in phosphate buffer pH 7.5 and centrifuged at 3000 rpm for ten minutes. The supernatants were collected and used for different analyses while the pellets were discarded.
2.6 Biochemical parameters studied 2.6.1 Biochemical parameters evaluated The oxidative stress biomarkers such as superoxide dismutase SOD , catalase CAT , glutathione peroxidase GPx , reduced glutathione, Glutathione-S-transferase GST , and malondialdehyde MDA were carried out on the rat heart homogenates, serum lipid profiles, serum cardiac troponin I, and creatine kinase MB were estimated via RX Daytona Automated Analyser Randox, UK in line with the manufacturer s guidelines. Also, tumor necrosis factor TNF -α and C-reactive protein were evaluated in heart homogenate using ELISA kits Biosource, USA at 450 nm.

Quantitative real-time PCR analysis
A known gram 0.5 µg of the newly harvested heart was inserted into 20 µL of Trizol 2 . Then, the total RNA of the organ was extracted through a cold solution of TRI reagent Zymo Research, USA by following the manufacturer s protocol. A spectrophotometer Jen-way UV-VIS spectrophotometer model 6305, UK was used to estimate the concentration of RNA at 260 nm . This was subsequently converted into cDNA via M-MuLV Reverse Transcriptase Kit by following manufacturer procedure. Hereafter, 25 µL of the solution comprising 2 µL cDNA, 2 µL primer, 12.5 µL Ready Mix Taq PCR master mix and 8.5 µL nuclease-free water were mixed.
The primer sequences used were indicated in Table 1.
Additionally, the quantification of the band images captured on the gel electrophoresis using 1.5 agarose was evaluated using Image-J software 13 .

Histological examination of the heart
A small portion 1 gram of the heart tissue obtained from each group was stored in a 10 formalin buffer. The tissues were then washed overnight, dehydrated with graded alcohols, and embedded in paraffin wax. For histological evaluation, 5 µm thick sequential section was deparaffinized and stained with hematoxylin and eosin H&E 14 .

Data analysis
A graph pad prism 5 GPP software was employed in this study. This was done through One-way ANOVA as well as Tukey multiple range post-hoc test. The data were articulated as the means of ten 10 replicates standard deviation SD , and a significant difference was fixed at p 0.05.

Aqueous extract of Sterculia tragacantha leaf sup-
presses oxidative stress biomarkers in the diabetic rats Diabetic control animals exhibited a remarkable decrease in SOD, CAT, GPx, GST and GSH levels when compared to other groups. However, diabetic rats placed on the two doses LDST and HDST of aqueous extract of Sterculia tragacantha leaf as well as MET revealed a significant p 0.05 upsurge in SOD, CAT, GPx, GST and GSH levels. Also, there was no significant p 0.05 difference in SOD, CAT, GPx, GST and GSH levels in normal control and diabetic rats administered HDST. In addition, diabetic control rats demonstrated a significant p 0.05 increase in MDA levels when compared to other groups, but at the end of the experimental trial, diabetic rats administered LDST, HDST, and MET exhibited no momentous difference compared to normal control Fig. 1 . Moreover, there was a substantial increase in activities of CAT, SOD, GPx, GST and GSH in diabetic rats administered HDST when compared to diabetic rats administered LDST.

Aqueous extract of Sterculia tragacantha leaf amelio-
rates hyperlipidemia in diabetic rats Diabetic control rats exhibited a significant p 0.05 rise in serum total cholesterol, triacylglycerols, and low-density lipoproteins LDL compared to other groups. There was no significant p 0.05 difference in total cholesterol, triacylglycerols, and low-density lipoproteins LDL of normal control and diabetic rats placed on HDST and MET. Diabetic control rats demonstrated a significant p 0.05 decline in

Aqueous extract of Sterculia tragacantha leaf inhibits in ammation and apoptosis in diabetic rats
Diabetic control rats demonstrated a significant p 0.05 rise in the level of cardiac troponin I and CKMB levels compared to other groups. Hence, diabetic rats treated with LDST, HDST, and MET revealed a significant p 0.05 reduction in cardiac troponin I and CKMB levels. Although diabetic rats treated with HDST and MET presented no significant p 0.05 difference with normal control rats as indicated in Fig. 3. Also, in diabetic control animals, there was a significant p 0.05 increase in the levels of Creactive protein when compared to other groups. Whereas, diabetic rats that received HDST indicate no significant p 0.05 difference compared with normal control rats. Furthermore, diabetic control rats show a significant p 0.05 increase in the level of TNF-alpha compared to other groups. This was drastically reduced in all the treated groups with no significant p 0.05 difference in diabetic rats given LDST, HDST and MET when linked to normal control rats.

Aqueous extract of Sterculia tragacantha leaf on rela-
tive gene expression of glucose transport 4 GLUT-4 , fatty acid-binding protein 3 FABP 3 , and urotensin II in diabetic rats There was a substantial reduction in GLUT-4 relative gene expression in the diabetic control group when linked to other groups. There was no significant p 0.05 difference in GLUT-4 relative gene expression of diabetic animals treated with HDST and MET when related to normal control rats Fig. 4 .
In Fig. 4, there was a noticeable reduction in relative gene expression of FABP3 in diabetic control animals when related to other groups. Also, there was a substantial difference in relative gene expression of FABP3 in diabetic rats treated with LDST, HDST and MET linked with both normal and diabetic control groups.
There was a noticeable upsurge in the relative gene expression of urotensin II in diabetic control rats over other groups. Also, the diabetic rats treated with LDST and HDST exhibited a significant p 0.05 difference when related to both normal and diabetic control rats Fig. 4 .

Aqueous extract of Sterculia tragacantha leaf on
heart histopathology examination in diabetic rats Figure 5 shows the histological examinations in the heart of diabetic rats administered aqueous extract of S. tragacantha leaves. There were severe degradation, congestion,

Discussion
The major approach of controlling diabetes mellitus is by lowering fasting blood glucose levels as well as all the associated complications. In this study, streptozotocin STZ was employed to mimic diabetes mellitus in experimental animals. A low dose of STZ was used to cause partial beta cell destruction, resulting in mild insulin deficiency 15 . This exhibited hyperglycemia in the animals, probably linked to the partial destruction of pancreatic beta-cell due to abnormal production of reactive oxygen species ROS by the diabetogenic agent used 12 . Hence, the administration of aqueous extract of Sterculia tragacantha ST leaf possibly demonstrated normoglycaemic potential on those diabetic animals as demonstrated in Figs. 1 to 5. This could be attributed to the antioxidant properties of ST leaf in protecting pancreatic β-cells against ROS, which encourages an upsurge in insulin secretion 16 .
One of the proposed mechanisms of action of this plant extract as anti-diabetic cardiomyopathy was its ability to counteract oxidative stress as indicated in Fig. 1. Persistent hyperglycemia stimulates the generation of ROS and reactive nitrogen species RNS in the heart mitochondria 17 . Surplus ROS support rises in lipid peroxidation MDA and subsequently changes membrane structure and enzyme activities 18 . In this study, diabetic rats presented momentously raised in MDA probably due to accumulation of free radicals which might lead to DNA fragmentation and protein damage of the heart tissue. This is in accordance with the previous study carried out by Alabi et al. 17 . However, this was ameliorated in diabetic rats that received ST leaf LDST and HDST probably due to its antioxidant ability. In another word, diabetic animals showed a reduction in cardiac GSH, designating its excessive usage in the redox-tackled cellular vicinity 19 . SOD, an enzyme involved in the dismutation of superoxide, CAT and GPx are enzymes that catalyze the breakdown of hydrogen peroxide free radical to water and oxygen 20 , and GST is a detoxification enzyme that functions in safeguarding cellular macromolecules from attack by reactive electrophiles 2 . The levels of all these enzymatic and non-enzymatic were noticeably increased in diabetic animals treated with the two doses of ST leaf, which were drastically reduced in diabetic rats, supporting their roles in scavenging the generated ROS due to STZ. This may be associated with ROS mopping up the ability of the extract. Thus, this is in accordance with the previous report by Al-Rasheed et al. 4 .
In addition, hyperlipidemia is an additional element of cardiac dysfunctions that can cause death in individuals with diabetes mellitus 21 . The findings obtained Fig. 2 showed that diabetic rats developed hyperlipidemia because of elevated levels of their lipid profiles with a reduction in HDL-C, presumably because of increment in the activation of free unsaturated fats from the fringe terminals. Hence, diabetic rats placed on LDST and HDST ame-liorate hyperlipidemia by increasing HDL-C, which makes it beneficial in the management of diabetic cardiomyopathy.
According to Frati et al. 22 , oxidative stress and inflammation are acknowledged in promoting diabetic cardiomyopathy via cardiac hypertrophy. In this study, persistent of hyperglycemia motivate cardiomyocyte damage as evidence in elevated serum CK-MB and cardiac troponin I Fig. 3 . Creatine kinase-MB CK-MB is an enzyme present mainly in the heart muscle cells, as one of the isoenzymes of creatine-kinase-CK. On the other hand, cardiac troponin I play an important role in the binding of actin in thin myofilaments to hold the actin-tropomyosin complex in order, it also averts myosin from binding to actin in the relaxed muscle 23 . One of the ways of detecting myofibrillar degeneration is by increased circulating CK-MB and cardiac troponin I 24 as noticed in this study. This may be linked to an increase in the level of inflammation as indicated in Fig. 1. However, diabetic rats administered LDST and HDST revealed a significant decline in the levels of these biomarkers probably connected to the anti-inflammatory ability of the extract as illustrated in Fig. 1. This is in line with the previous report by Safhi et al. 25 .
Suzuki et al. 26 stated that cardiac inflammation, apoptosis, and fibrosis are all important factors in DCM. The persistent increase in ROS generation in STZ-induced diabetes animals may be responsible for an increase in the level of circulating CRP and TNF-α Fig. 3 . C-reactive protein CRP serves as innate immunity, as well as an early defense system against infections in the human body system 27 . Tumour Necrosis Factor-alpha TNF-α , is an inflammatory cytokine produced by macrophages during acute inflammation trigger by ROS leading to necrosis 28 . Remarkably, diabetic rats maintained on LDST and HDST showed a significant reduction in serum CRP and TNF-α levels. Therefore, this corroborates the anti-inflammatory ability of the plant extract used as previously reported in Fig. 1.
Thorens and Mueckler 29 reported that mammalian cells consume glucose, mainly for energy generation, via glucose transporter proteins. GLUT-4 is a glucose transporter responsible for glucose uptake into the cardiac muscles. In the diabetes mellitus condition, the level of GLUT-4 is always hindered in cardiac muscles which encourages hyperglycemia in the bloodstream 30 . This was witnessed in this study Fig. 4 , however, the administration of LDST and HDST into the diabetic animals was able the increase the level of GLUT-4 gene expression and possibly promote glucose assimilation into the cardiac muscles.
Urotensin-II gene U-II is a well-known factor in vascular contraction in mammals, especially in the heart 31 . The U-II plays an important role in insulin resistance, impaired glucose tolerance, impaired glucose transport to muscles through GLUT-4, etc. 32 . Therefore, over-expression of the cardiac U-II gene is an indicator of DCM as shown in Fig. 4, also, its over-expression can be linked to its noticeable decreased in GLUT-4 levels as indicated in Fig. 4 . Interestingly, diabetic animals maintained on LDST and HDST were able to normalize the level of U-II, probably due to an increase in GLUT-4 relative gene expression as demonstrated by this extract. This is in accordance with the report of Jafria et al. 30 .
According to Kusudo et al. 33 , fatty acid-binding proteins 3 FABP3 are known as small cytosolic proteins, belonging to a superfamily of lipid-binding proteins. FABP3 expressions are present in series of cells, particularly the heart cells. FABP3 is involved in fatty acid FA uptake, transport, esterification, and β-oxidation for energy generation, particularly in the heart muscles 34 . Hence, in this study, the significant decrease in FABP3 expression in diabetic control animals may be associated with over usage of this protein in FA uptake due to non-useful of the available glucose for the body system. But at the end of the experimental trial, the LDST and HDST were able to boost the expression of this protein, which may be associated with their ability to make glucose available into the cells and render a decrease in FA uptake Fig. 4 .
Moreover, the histological examination of the diabetic control heart Fig. 5 showed myocardium degeneration as well as improved collagen deposition. This may be linked to oxidative stress, due to persistent hyperglycaemia experienced in this study. This is in line with the early work described by Al-Rasheed et al. 16 . In another vein, cardiomyocytes, as well as inflammatory cells, encourage fibrosis in the animal s diabetic control heart due to the release of cytokines and profibrotic factors 25 . However, administration of LDST and HDST noticeably reduced the synthesis of collagen and fibrosis, presumably via its antioxidant booster ability which led to a reduction in heart inflammation in diabetic animals. Therefore, the administration of ST inhibited histological alterations and deposition of collagen in the heart of diabetic animals. Added to this, the proposed mechanism of action of ST is illustrated in Fig. 6.

Conclusion
It can be presumed from this study that aqueous extract of Sterculia tragacantha leaf ameliorated hyperlipidemia, oxidative stress biomarkers by increasing the antioxidant enzyme activities and decrease in lipid peroxidation, enhanced CK-MB, CRP, cardiac troponin I, TNF-α, and urotensin-II levels, and improved GLUT4 and FABP3 expressions, supported by histological examination. However, Fig. 6 Proposed mechanism of action of diabetic cardiomyopathy rats administered aqueous extract of S. tragacantha leaves.