Apigenin-Loaded Solid Lipid Nanoparticle Attenuates Diabetic Nephropathy Induced by Streptozotocin Nicotinamide Through Nrf2/HO-1/NF-kB Signalling Pathway.

BACKGROUND: Apigenin is known to have a broad-spectrum efficacy in oxidative stress and conditions due to inflammation, although weak absorption, fast metabolic rate and a fast elimination (systemic) limit the pharmacological efficacy of this drug. Hence, we propose the usage of highly bioavailable Apigenin-solid lipid nanoparticles (SLNPs) to recognize such limitations. The defensive function of Apigenin-SLNPs on renal damage induced by streptozotocin (STZ) in animals was studied.
MATERIALS AND METHODS: We initially injected the rats with 35 mg kg-1 streptozocin intraperitoneally, and after 7 days, the rats were then injected 150 mg kg-1 of metformin intragastrically followed by a once-daily intragastric dose of Apigenin-SLNP (25 or 50 mg kg-1) for a continuous period of 30 days. We then measured the level of insulin and blood glucose, superoxide dismutase, catalase and malondialdehyde in the tissues of the kidney. We also observed messenger-RNA expression of Interleukin-1β, Interleukin-6 and Tumor Necrosis Factor-alpha in renal tissue through RT-PCR technique. Moreover, H&E staining and Western blotting observed the histopathological variations and protein expression of nuclear factor erythroid 2-related factor 2/heme oxygenase/Nuclear Factor-κB signaling pathway, respectively.
RESULTS: An enhancement in the expressing of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 and a suppression in the expression of Nuclear Factor-κB occurred due to Apigenin-SLNPs treatment, which was a result of the protective mechanism of Apigenin-SLNPs which is because of not only its anti-inflammatory function (by inhibition of release of inflammatory factors) but also their anti-oxidant activity (through reduction of lipid peroxidation production).
CONCLUSION: We found that a protective effect on diabetic nephropathy was shown due to Apigenin-SLNPs, in rats induced with streptozocin maybe through the pathway of nuclear factor erythroid 2-related factor 2/heme oxygenase-1/Nuclear Factor-κB.

Introduction

Diabetes mellitus (DM) is a multi-dimensional metabolic disorder which is associated with a deficit, wherein, the glucose utilization of the body and insulin homeostasis maintenance are affected. A chronic ruination, deterioration and an eventual breakdown of the organs mainly the eye, kidney, nerves and the cardio vascular system is a result of a constant hyperglycaemia. Globally, there are 366 million DM patients and the numbers of new cases are predicted to double up by the year 2030.1 Diabetic nephropathy (DN) is a common disorder that occurs because of both type 1 and type 2 DM and affects DM-associated mortality drastically. It is also responsible for renal dysfunction in high-income countries. The clinical assessment of DN is done through a five-stage criterion, wherein each stage features unique sets of functional and clinical alterations along with alterations in the standard renal function markers.2 According to various studies, insulin resistance and the state of DM govern the production of oxidative stress to a large extent. Recent studies have suggested DN to be an inflammatory process and the progression of which can lead to implication of immune cells, even though the heamodynamic and metabolic factors are most common causative factors of DN.3–5 Mesangial expansion, alterations of extracellular matrix, tubulointerstitial renal fibrosis, and glomerulosclerosis are the leading pathological conditions of DN. This is due to the diverse adverse effects caused by chronic use of hyperglycemic drugs, and hence, plant sources with negligible adverse effects are now being considered in the development of hyperglycemic drugs.6 Phytoconstituents with a persuasive antioxidant property have proved to be a breakthrough remedy against DM. Also, they have been highly considered as source of biologically active substance (antioxidants, anti-hyperglycemics and anti-hyperlipidaemics).7

Apigenin is a flavonoid naturally present in tea, berries, fruits, and vegetables. These have had various biological functions, like antioxidant and anti-inflammatory activity.8 Apigenin was reported to be playing a protective role in oxidative-related disorders like CVD and neurology-related diseases. Apigenin alleviates myocardial toxicity by modulating oxidative stress and inflammation in myocardial injury model reactive oxygen species (ROS) and  malondialdehyde (MDA) decreased significantly in rat acrylonitrile-induced subchronic sperm injury.9–11 Pharmacological studies have confirmed that Apigenin markedly inhibited glomerular mesangial cell proliferation, glomerulus hypertrophy, and extracellular matrix development and aggregation in DN patients and mice. Apigenin plays a multi-target role in DN treatment. Its low solubility, short half-life, low renal concentration, and minimal bioavailability hamper Apigenin uses.12 Therefore, in laboratory animals, this research aimed to examine the attenuating effects of Apigenin-solid lipid nanoparticles(SLNPs) in diabetic nephropathy caused by streptozotocin (STZ) nicotinamide.

Materials and Methods

Chemicals

We obtained Apigenin and STZ from Sigma. Glucose, UA, Creat. and commercial kits were provided by Jiancheng Bioengineering Institute. Rest of the chemicals and reagents in use were of analytical grade.

Preparation of Apigenin-SLNPs

We used microemulsification method to prepare SLNPs. We then briefly placed a mixture of 45.45% Tween 80, 0.58% PLPC, and water in a beaker which was then subjected to heat to a temperature to attain the lipid-melting point. We also melted Lipid (7.27%) at a temperature of 82°C to 85°C separately. We then added Apigenin (25 mg) aqueou phase containing Tween 80, later we dropped the hot aqueousemulsifier mix into the lipid melt all at once. This was done under magnetic stirring for obtaining a clear micro-emulsion. We then transferred hot microemulsion in cold water (~2°C) of an equal amount. This process was carried out under mechanical stirring at a speed of 5000 rpm for a time period of 1.5 hrs. The SLNPs are formed in aqueous medium by crystallization of the hot droplets of lipid that are present in the microemulsion. The aqueous  SLNP dispersion that was prepared was refrigerated until further analysis.13

Characterization Studies of Apigenin-SLNPs

Nano-ZS ZEN 3600 measured the polydispersion (PDI), zeta potential, mean size, and size distribution of Apigenin-SLNPs using the dynamic light scattering technique. We measured the scattering frequency at 90° and 25° angles. TEM investigated on the size of the Apigenin-SLNPs. We examined the morphology of the Apigenin-SLNPs by using SEM. We then viewed SEM images of Apigenin-SLNPs by a Scanning Electron Microscope.

Evaluation of Apigenin-SLNPs Encapsulation Efficiency

We determined the nanoformulation encapsulation-efficiency as the percent of Apigenin trapped in the nanoparticles. We then dissolved 25 mg of Apigenin nanoparticles with 1 mL of 90% methanol, further subjected to sonication for 5 mins in order to cause disruption of the nanoparticles and release encapsulated Apigenin. The resulting solution at 10,000xg was centrifugated and we then gathered the supernatant. Supernatant absorption was read to quantify Apigenin at 425 nm in a spectrophotometer.14

In vitro Drug Release

Apigenin-SLNPs were screened with a dialysis bag technique for the in vitro discharge profile. In short, a 25 mg Apigenin-SLNPs in PBS (2 mL) were suspended and moved to a dialysis bag (molecular weight 12,000–14,000Da). We secured the pocket with pins which were held in a bottle at 50 mL phosphate buffer saline (pH 7.4) with 1% of polysorbate 80 under steady agitation at a speed of 100 rpm. One millilitre PBS has been removed at 1 hr intervals and spectrometric tests of Apigenin have been conducted at 425 nm. After each removal, we introduced 1 mL new phosphate buffer saline in the container to preserve fluid circumstances.15

Experimental Design

Animals

We acquired adult rats (male) weighing 180 to 200 gm from the animal house of Zhengzhou Central Hospital, China. We then allowed them to adapt to a new environment for 5 days. We maintained the animals in a 12 hr light-dark natural cycle at an ambient temperature of 24 ± 1°C. The animals were given a standard diet and water ad lib. We obtained approval from Zhengzhou University Committee on Animal Care, also we followed all the procedures as per the legislation of China on the use and care of experimental animals and the guidelines given by Institute for Experimental Animals of Zhengzhou Central Hospital.

Induction of Diabetes

To prepare diabetic models, we injected the rats with 35 mg kg−1 of STZ intraperitoneally, which was made in 0.1 M citrate buffer with a pH of 4.4. Alongside, we administered the same amount of citrate buffer to the control group (n=10). We collected the blood sample from the orbital venous plexus. Post 7 days of streptozocin, we estimated their levels of blood glucose with the help of a kit. For further study, we only selected animals who had a blood glucose level ≥11.1 mmol L−1. We randomly divided the rats with DM into four groups having 10 rats in each group. The groups were: Streptozocin group, Streptozocin + Metformin (150 mg kg−1) group, Streptozocin + Apigenin-SLNPs (25 mg kg−1) group, and Streptozocin + Apigenin-SLNPs (50 mg kg−1) group. On confirmation of DM in the rats, we administered them with Metformin (150 mg kg−1), Apigenin-SLNPs (25 mg kg−1) or Apigenin-SLNPs (50 mg kg−1) intragastrically, once daily for 30 days consecutively. Control and Streptozocin group were simultaneously given equal volume of distilled water. When the experiment period was completed, we sacrificed the rats after the rats were given anesthesia (urethane 20%). We obtained blood samples, which were kept for 20 mins at ambient temperature, meanwhile the clots formed. The samples were further subjected to centrifugation at a speed of 3000 rpm for a period of 10 mins. For histopathological examination, we excised three renal tissues and then fixed in neutral buffered formalin (10%). Remaining tissues of the kidney were preserved at −70°C till further usage.16,17

Measurements of Blood Glucose and Insulin

Determination of insulin and blood glucose in the serum was done by the aid of glucose and ELISA kit as per the protocol of the manufacturer.

Superoxide Dismutase (SOD), Catalase (CAT), and MDA Measurement in Renal Tissue

We determined the renal tissues’ Catalase, superoxide dismutase, and malondialdehyde levels with the help of purchased test kits purchased. We carried out all the procedures in accordance to the manual.18

qRT-PCR

Total RNA were subjected to isolation from kidney samples we prepared with the use of guanidinium thiocyanate reagent. By RT, complementary DNA was synthesized in accordance to the protocol of the manufacturer. We performed qRT-PCR with a std. Synergy Brands, Inc. green Polymerase Chain Reaction kit, we also performed a PCR amplification which was gene specific by the use of ABI 7300. We calculated levels of relative gene expression by the use of 2-ΔΔCt methodology after normalizing the levels of messenger RNA of GAPDH. Primers used for real-time PCRs are as follows: Tumor Necrosis Factor-alpha, Fwd: 5ʹ-ACTTTGGAGTGATCGGCCCC-3ʹ; Rev: 5ʹ-TTCTGTGTGCCAGACACCCTA-3ʹ, IL-6, Fwd: 5ʹ- CCTTCTCCACAATACCCCCAGG-3ʹ; Rev: 5ʹ- TGTGCCCAGTGGACAGGTTT-3ʹ and IL-1β, Fwd: 5ʹ- ACCTGAGCTCGCCAGTGAAAT-3ʹ; Rev: 5ʹ-ACCCTAAGGCAGGCAGTTGG-3ʹ, GAPDH, Fwd: 5ʹ- TGGGGTGATGCAGGTGCTAC-3ʹ; Rev: 5ʹ- GGACACGGAAGGCCATACCA-3ʹ.19

Histopathological Changes

We harvested the kidney tissue longitudinally and was fixed in paraformaldehyde (4%), embedded with paraffin and sliced into sections (4-µm) and then stained with H&E staining, we then observed this under an optical microscope. We examined deep coronal section with the help of a microscope, which was then graded as per the magnitude of damage which was based on the % involvement of the kidneys. We graded the damage quantification from ten areas corresponding to the renal PT by use of following parameters: tubular cell necrosis, cytoplasmic vacuole formation, hemorrhage, and tubular dilatation.20

Western Blot

Western Blot

We performed western blot as mentioned previously. Shortly, 150 μg protein aliquots from the supernatant of the tissue of the kidney were run on SDS-PAGE (10%). We blocked bull serum albumin (3%) in 0.2% to 0.4% TBST for 1 hr. After doing so, we incubated the membranes at 4°C overnight with primary Ab against active TAT-14 Peptide, Heme Oxygenase-1, and NFKB and later with alkaline phosphatase-conjugated secondary Ab. We developed the membranes by 5-b-romo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. We stained the blots with an anti-β-actin Ab. The protein levels were then normalized concerning β-actin band density. We measured the Ag-Ab products by Thermo Scientific Super Signal West Pico Chemiluminescent Substrate. We analyzed the result with a Fluor Chem system.21

Statistical Analysis

We expressed the data as mean ± SEM. We used ANOVA to evaluate the difference between the groups by using Tukey’s multiple comparison test (significance p value < 0.05).

Results

Characterization of Prepared Apigenin-SLNPs

SEM, TEM, and light dynamics were used to execute the Morphology and Distribution of the Apigenin-SLNPs. The prepared Apigenin-SLNPs are small, compact spheres with an average size of 152.7 ± 7.04 nm of particles as shown in Figures 1 and 2 and have a zeta potential of 52.18±3.9 mV and 0.692 PDI. Apigenin-SLNP’s encapsulation performance was 78.90%. Figure 2B shows the quantity of the released drug against time for Apigenin-SLNPs. The average release rate of a medication up to 24 hrs is 71.52% with a rapid release of 34.28% in 5 hrs. Thereafter, the percentage release over 24 hrs was steadily increased.

Figure 1

(A) SEM micrographs of the Apigenin-SLNPs, (B) TEM image of Apigenin-SLNPs.

Figure 2

(A) DLS analysis of the Apigenin-SLNPs, and (B) in vitro release profile of Apigenin-SLNPs.

Apigenin-SLNPs Effect on Insulin and Blood Glucose

Table 1 shows, insulin and blood glucose levels in diabetic animals induced by STZ were suggestively greater as compared to controls (p value<0.001). Comparatively, Apigenin-SLNP (25 and 50 mg kg−1) and metformin therapies effectively reduced level of glucose in the blood and increased insulin levels in rats given Streptozocin (significance p<0.05, <0.01, and <0.001). Such findings show that diabetic rats are induced by STZ, and Apigenin-SLNPs exhibited anti-diabetic properties.

Table 1

Effects of Apigenin-SLNPs on Blood Glucose and Insulin

Treatment Groups	Blood Glucose (mmol/L)	Serum Insulin (ng/mL)
Control	06.83±1.93	09.04±1.73
STZ	13.84±1.82z	4.92±2.83z
Metformin	07.02±1.03c	10.92±1.04c
STZ+Apigenin-SLNPs (25)	10.55±1.78a	10.05±1.92b
STZ+Apigenin-SLNPs (50)	07.14±1.21c	7.60±1.21c

Notes: Values are expressed as means ± SEM. Compared with control: zP < 0.001; compared with STZ: aP < 0.05, bP < 0.01 and cP < 0.001.

Abbreviations: Apigenin-SLNPs, Apigenin-solid lipid nanoparticles; STZ, streptozotocin.

Apigenin-SLNPs Effect on Oxidative Stress

Lipid peroxidation was measured by measuring SOD, catalase as well as MDA. Table 2 shows the stimulation of streptozocin markedly reduced superoxide dismutase and catalase activities (p value <0.001). Thus, Apigenin-SLNPs (25 and 50 mg kg−1) as well as metformin therapies were successful in restoring superoxide dismutase and catalase levels (significant p<0.05, <0.01, and <0.001). Furthermore, STZ treatment revealed a remarkably strong MDA amount (p<0.01). On the opposite, prescribing Apigenin-SLNPs (25 and 50 mg kg−1) and metformin dramatically reduced MDA quality (p value <0.05, <0.01, and <0.001). Our findings have shown these Apigenin-SLNPs could decrease oxidative-stress STZ-induced diabetic rats.

Table 2

Effects of Apigenin-SLNPs on MDA, SOD and CAT

Treatment Groups	MDA (nmol/mg prot)	SOD (U/mg prot)	CAT (U/mg prot)
Control	1.93±0.04	79.61±3.28	17.27±1.72
STZ	4.89±0.18z	20.88±1.04z	6.03±1.00z
Metformin	1.98±0.49c	75.81±3.05c	16.77±1.32c
STZ+Apigenin-SLNPs (25)	3.05±0.08a	58.26±3.29b	10.04±1.72a
STZ+Apigenin-SLNPs (50)	2.02±0.50c	72.90±3.81c	14.28±1.04b

Notes: Values are expressed as means ± SEM. Compared with control: zP < 0.001; compared with STZ: aP < 0.05, bP < 0.01 and cP < 0.001.

Abbreviations: Apigenin-SLNPs, Apigenin-solid lipid nanoparticles; CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase; STZ, streptozotocin.

Effect of Apigenin-SLNPs on Interleukin-6, Interleukin-1β, and Tumor Necrosis Factor-Alpha mRNA Expression

For STZ-administered rats, Interleukin-6, Tumor Necrosis Factor-alpha and Interleukin-1β mRNA expression were expressively up-regulated (p<0.001) in contrast with control rats. Thus, Apigenin-SLNPs (25 and 50 mg kg−1) and metformin therapies are significantly down-regulated (p value< 0.05, < 0.01, and < 0.001) Interleukin-6, Tumor Necrosis Factor-alpha and Interleukin-1β mRNA expression relative to diabetic rats induced by STZ (Figure 3).

Figure 3

Effect of Apigenin-SLNPs on the expression of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the kidney of STZ treated rats.

Apigenin-SLNPs Effect on Renal Tissue Histopathology

Apigenin-SLNPs defensive function in physiological dysfunction, H&E staining was assessed. Histological analysis of control-group renal tissue revealed normal cell architecture. Conversely, diabetic rat kidneys showed extreme tubular necrosis, mild glomerular dilation, and interstitial inflammation. Nonetheless, Apigenin-SLNPs and metformin attenuated the extent of renal damage. Analytical results showed that Apigenin-SLNPs strengthened the histopathological status of streptozocin-induced DN (Figure 4).

Figure 4

Apigenin-SLNPs effect on histopathological changes of renal tissue.

Apigenin-SLNPs Effect on Proteins Expression of TAT-14 Peptide, NRKB and Heme Oxygenase-1

In the antioxidant reaction, TAT-14 Peptide/heme oxygenase-1 has a crucial role. To examine if TAT-14 Peptide and heme oxygenase-1 expression are intertwined with the defensive effects of Apigenin-SLNPs on STZ-induced oxidative damage to the kidneys. As predicted, NF-kB protein expressions have increased substantially, whereas diabetic rats caused by STZ expressions of Nrf2 and HO-1 have been substantially reduced relative to control (P<0.001). Thus, Apigenin-SLNPs (25 and 50 mg kg−1) and metformin therapies effectively reduce controlled protein expression of NF-πB and TAT-14 Peptide and heme oxygenase-1 regulated expression in support of diabetic rats caused by STZ (p value <0.05, <0.01, and <0.001), as seen in Figure 5.

Figure 5

Effect of Apigenin-SLNPs on the protein’s expression of Nrf2, Nf-kB and HO-1 in the kidney of STZ treated rats.

Discussion

Apigenin was encapsulated into SLNP through the microemulsification method. We placed 45.45% Tween 80, 0.58% PLPC, and water together in a beaker which was subjected to heat till it attained lipid melting temperature. We also melted 7.27% lipid at a temperature of 82°C to 85°C separately. We then added Apigenin to the aqueous  phase that contained Tween 80, after which we dropped the hot aqueous  emulsifier mixture into the lipid melt under continuous magnetic stirring in order to get a microemulsion which is visibly clear and is more advantageous than using Apigenin alone. An improvement in the biodistribution, elevated sensitivity and specificity, and reduced pharmacological toxicity can be attained by modification to nanoparticle. The obtained nanoparticles in our study had a particle size of 152.7 ± 7.04 nm, the small particle size may increase its antioxidant and anti-inflammatory properties.

DM is a common disorder related to metabolism and has a partial relation to the lipid peroxidation derangements. Pancreatic cell apoptosis is caused due to streptozocin, and hence makes it an experimental model for inducing T1DM.22,23 In our study, the protective effect of Apigenin-SLNPs on the kidney injury of streptozocin-induced diabetic rats is by its anti-oxidative and anti-inflammatory properties.24 Streptozocin-induced diabetic model was confirmed to be a well-established model due to elevated levels of blood glucose and insulin. Also, the levels of blood glucose and insulin were decreased by Apigenin-SLNPs treatment which indicated it to possess a protective shield against DM.25,26 At the same time, the study of histopathology of streptozocin-stimulated renal sections showed grave degeneration of the tubules (necrosis), glomerular dilatation (moderate), thickening of the vascular wall and inflammation of the interstitium. These alterations were reduced by Apigenin-SLNPs. The results on the whole suggested the efficacy of Apigenin-SLNPs over streptozocin-challenged diabetic rats.

An imbalance between oxidants and antioxidants is known to cause Oxidative stress. The ROS which was accumulated could have an interaction with fatty acids (polyunsaturated) leading to lipid peroxidation formation in the renal tissues. This could ultimately result in damage and toxicity.27 The fact that oxidative stress is the major factor for DM-related complications including DN is acknowledged widely.28,29 The membrane fatty acid (polyunsaturated) is degraded by ROS and leads to production of 4-HNE and MDA, which is an unstable aldehyde that could form a covalent protein adduct which is a trait of oxidative stress in tissue injuries. Superoxide dismutase and catalase are free radical scavenging enzymes, which act as the first defense line against oxidative damage in mammals. The oxidation/reduction/conversion of SOD radicals (O2−) to molecular O2 and hydrogen peroxide is catalyzed by superoxide dismutase. There is evidence of existence of a close association of superoxide dismutase and malondialdehyde with DN. Catalase is considered to be a prominent antioxidant in the kidneys that are responsible for the elimination of hydrogen peroxide and also guards the tissues from the reactive OH− radicals.30,31 Studies in the past have shown an accelerated kidney injury in DM due to catalase deficiency due to deficiency of the peroxisome. The result of our study confirms the role of Apigenin-SLNPs in an increase in activity of superoxide dismutase and catalase and also reduced levels of malondialdehyde in the renal tissues of rates stimulated with streptozocin.

Besides, the anti-inflammatory effect of Apigenin-SLNPs was validated from the mRNA expression of cytokines from renal tissues. mRNA level of Interleukin-6, Tumor Necrosis Factor, and Interleukin-1β were highly stimulated in renal tissues of diabetic rats, whereas Apigenin-SLNPs treatment decreased the level as that of normal rats. Studies have shown that Interleukin-6 has a major function in leukocyte recruitment, apoptosis, and activation of T-lymphocyte.32,33 Also, Tumor Necrosis Factor is known to stimulate neutrocytes for transcribing and releasing of cytokines and chemokines biosynthesis.34 Inhibiting Tumor Necrosis Factor-alpha, Interleukin-1β, and Interleukin-6 release may decrease inflammation. Nuclear Factor-κB has a major role in the nephropathy (pathophysiologically) in rats with DM with inflammation and oxidative stress. Activation of Nuclear Factor-κB expression is positively related to the expression of Tumor Necrosis Factor-alpha, Interleukin-1β, and Interleukin-6. The property of heme oxygenase-1 to catabolize free heme and also to produce CO is responsible for its inherent anti-inflammatory property by up-regulation of Interleukin-10. Nuclear factor erythroid 2-related factor 2 is an important regulator of antioxidative defence pathway which can be activated by diabetes and DN.35,36

An earlier study has shown that the enhancement of Nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway has shown an anti-apoptosis activity and also that activating Nuclear factor erythroid 2-related factor 2 could lead to induction of heme oxygenase-1 expression.37 From the current study, we inferred that there was a down-regulation in the expression of Nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 whereas, there was an up-regulation in the expression of Nuclear Factor-κB in rats with DM.10 Treatment with Apigenin-SLNPs improved Nrf2 and HO-1 expression and reduced NF-kB activity, suggesting that Apigenin-SLNP protection is based on its anti-inflammatory activity by decreasing the development of LPO by preventing the pro-inflammatory factors release and its anti-oxidant activity, which is likely dependent on controlling activated IL-6, IL-1β, TNF-α and NF-κB through Nrf2/HO-1 signaling pathway on DN induced by STZ.

Conclusion

Our results showed that Apigenin-SLNPs possessed a protective effect by reducing the OS and inflammation which might be through the Nuclear factor erythroid 2-related factor 2/heme oxygenase-1/Nuclear Factor-κB signaling pathway. More studies are needed for the exploration of clinical application of Apigenin-SLNPs.