GANE can Improve Lung Fibrosis by Reducing Inflammation via Promoting p38MAPK/TGF-β1/NF-κB Signaling Pathway Downregulation.

There is a trend to use nanoparticles as distinct treatments for cancer treatment because they have overcome many of the limitations of traditional drug delivery systems. Gallic acid (GA) is an effective polyphenol in the treatment of tissue injuries. In this study, GA was loaded onto niosomes to produce gallic acid nanoemulsion (GANE) using a green synthesis technique. GANE's efficiency, morphology, UV absorption, release, and Fourier-transform infrared spectroscopy (FTIR) analysis were evaluated. An in vitro study was conducted on the A549 lung carcinoma cell line to determine the GANE cytotoxicity. Also, our study was extended to evaluate the protective effect of GANE against lipopolysaccharide (LPS)-induced pulmonary fibrosis in rats. GANE showed higher encapsulation efficiency and strong absorption at 280 nm. Transmission electron microscopy presented a spherical shape of the prepared nanoparticles, and FTIR demonstrated different spectra for the free gallic acid sample compared to GANE. GANE showed cytotoxicity for the A549 carcinoma lung cell line with a low IC50 value. It was found that oral administration of GANE at 32.8 and 82 mg/kg.b.w. and dexamethasone (0.5 mg/kg) provided significant protection against LPS-induced pulmonary fibrosis. GANE enhanced production of superoxide dismutase, GPx, and GSH. It simultaneously reduced the MDA level. The GANE and dexamethasone, induced the production of IL-4, but suppressed TNF-α and IL-6. On the other hand, the lung p38MAPK, TGF-β1, and NF-κB gene expression was downregulated in rats administrated with GANE when compared with the LPS-treated rats. Histological studies confirmed the effective effect of GANE as it had a lung-protective effect against LPS-induced lung fibrosis. It was noticed that GANE can inhibit oxidative stress, lipid peroxidation, and cytokines and downregulate p38MAPK, TGF-β1, and NF-κB gene expression to suppress the proliferation and migration of lung fibrotic cells.

Introduction

Pulmonary fibrosis is a group of lung diseases in which collagen deposition and fibroblast proliferation cause normal tissues to be replaced with a scar one.[1] Fibrotic illness can be caused by heavy metal dusts, some chemotherapeutics, silica, malachite, dust, and exposure to radiation.[2−4]

Immune response,[5] circulating immune cells,[6] and accumulated fibroblasts[7] are also involved in the development of pulmonary fibrosis. On the other hand, pulmonary fibrosis and assess disease severity can evaluated by interleukin levels.[8,9]

Also, oxidative stress can induce lung inflammation and aggravate the elevation of pulmonary fibrosis and eventually lead to bronchitis and emphysema.[10]

To induce pulmonary fibrosis, rats are given triggering agents such as lipopolysaccharide.[11]

Lipopolysaccharide is a component of the Gram-negative bacterial cell wall found in atmospheric pollutants.[12] When the bacteria infiltrate an organism’s lung, the lipopolysaccharide stimulates macrophages, neutrophils, and epithelial cells to release inflammatory factors, causing lung tissues and airways to inflammate.[13]

Polyphenols are a large group of phytochemicals that are naturally found in plants and beverages including fruit, vegetables, cocoa, cereal, tea, wine, beer, and coffee and are characterized by their hydroxylated phenyl groups.[12] One of the most common phenolic acids; gallic acid (Figure ), a flavoring agent and preservative in the food industry,[14,15] has been reported for its biological and pharmacological activities,[16−21] cardioprotective,[22,23] neuroprotective[24] gastroprotective,[25,26] and metabolic disease prevention.

Figure 1

Chemical structure of gallic acid.

Nanoform of drugs has led to several applications in cancer diagnosis and treatment,[27] drug formulation,[28] biomarker mapping,[27] targeted therapy, molecular imaging, and development of nanomaterials.[29−32]

Niosomes have become popular in drug delivery due to their unique properties.[33,34] It is spherical in shape and consists of non-ionic surfactants at variable molar ratios with cholesterol. Niosomes can deliver insoluble and hydrophilic agents to the part to be treated in a minimum dose to decrease the side effects, enhance the therapeutic effects, increase the stability of the drug, prolong, and enhance drug absorption into the target area.[35−41]

As a continuation of our interesting research in evaluation of therapeutic potential drugs of medical importance,[42−44] we report herein, a facile route to prepare a new nano-formula for gallic acid and evaluate its preventive efficacy against LPS-induced lung fibrosis in rats.

Results

Encapsulating Efficiency

In the present study, niosomes reveal a high efficiency for encapsulating gallic acid (67 ± 3.0 %).

UV Spectrophotometry of Gallic Acid

In this work, we synthesized niosome nanocarriers encapsulating gallic acid (gallic acid-niosomes). The UV spectrum of free and nanocarriers is shown in Figure a. They have a strong absorption at 280 nm. Transmission electron microscopy (TEM) micrographs showed the spherical shape of the prepared nanocarriers (Figure b). The size of gallic acid-niosomes is 58–70 nm.

Figure 2

UV spectrum of free gallic acid and niosomes encapsulating gallic acid (a) TEM image of the niosomes encapsulating gallic acid nanoparticles (b).

In-Vitro release

The release GA from the dialysis bag was rapid and reached equilibrium within 6 h (Figure ) due to its particles were unrestricted and had freedom of movement. The release of GA from niosomes took a longer time interval (Figure ) since GA was located within the niosomes.

Figure 3

Sustained release of gallic acid.

FTIR

Figure presents the mean Fourier-transform infrared spectroscopy (FTIR) spectra of free GA and GANE samples. Remarkably, the free GA sample demonstrates different spectra compared to the GANE. GA has a peak at 1701.87 cm–1 , which relates to a carbonyl group, and a band at 3295–3400 cm–1 corresponding to the phenolic O–H stretch group and the peak at 1030.77 cm–1 to the benzene ring. There are some differences in the GANE spectrum compared with that of free GA at the region between 3550 and 3200 cm–1 that corresponding to O–H group. This dissimilarity indicates that the incorporation of gallic acid into niosomes adds −OH groups to the aromatic rings in the spectrum of the GANE. The FTIR spectrum demonstrates that GA may be incorporated into niosomes.

Figure 4

Average spectra of samples after area normalization for free gallic acid, and the niosomes encapsulating gallic acid nanoparticles.

GANE Cytotoxicity on A549 Lung Carcinoma Cell Line

The results reported in Table and Figure show that the incubation of GANE at different consternations (31.25, 62.50, 125, 250, 500, and 1000 μg mL–1) with A549 lung carcinoma cell line resulted in viability % of 98.23, 71.47, 28.13, 9.31, 5.39, and 5.09, respectively, and toxicity % of 1.76, 28.52, 71.86, 90.68, 94.60, and 94.90, respectively. The IC50 value of the GANE against A549 cells was 94.47 μg mL–1.

Table 1

Determination of GANE Cytotoxicity on A549 Lung Carcinoma Cell Line (MTT Protocol)at

conc μg/mL	O.D			mean O.D	St.E	St.D	main viability %	main toxicity %	IC50
DMSO (0.1%)	0.326	0.353	0.341	0.34	0.008	±0.01	100	0
1000	0.017	0.018	0.017	0.017	0.001	±0.01	5.09	94.9	94.47
500	0.018	0.019	0.018	0.018	0.001	±0.01	5.39	94.6
250	0.020	0.038	0.037	0.032	0.006	±0.01	9.31	90.7
125	0.089	0.105	0.093	0.096	0.005	±0.01	28.1	71.9
62.5	0.239	0.236	0.254	0.243	0.006	±0.01	71.4	28.5
31.25	0.333	0.326	0.343	0.334	0.005	±0.01	98.2	1.77

Data shown are mean optical density ± SD of number of 3 observations within each treatment. O.D. = optical density, mean O.D. = average optical density, St. E. = standard error, St.D. = standard deviation, IC50 = half-maximal inhibitory concentration.

Figure 5

Effect of GANE cytotoxicity on A549 lung carcinoma cell at different concentrations.

GANE Cytotoxicity in Vivo

The results reported in Table present the oral administration of GANEs in doses 800, 1200, 1600, 2000, 2400, and 2800 mg/kg b.w. Yielded mortalities 0, 2, 5, 8, 9, and 10 respectively. LD50 dose of GENE s was 1640 mg/kg b.w.

Table 2

Determination of LD50 of GANE Given Orally in Adult rats

group number	dose (mg/kg)	no. of animals/group	no. of dead animals	(Z)	(d)	(Z.d)
1	800	10	0	1.0	400	400
2	1200	10	2	3.5	400	1400
3	1600	10	5	6.5	400	2600
4	2000	10	8	8.5	400	3400
5	2400	10	9	9.5	400	3800
6	2800	10	10	0	00	6950

.

.

Effect of GANE on Plasma TC, TG, and HDL-C Levels

Figure shows plasma total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL)-C levels. Oral administration of LPS (200 μg/kg b.w.) led to a significant increase of TG level to 37.94%, and significant (P < 0.01) decrease in plasma TC and HDL-C level to 34.64 and 53.51%, respectively, compared to the control. Treatment of animals by the GANE (32.8 mg/kg.b.w.) significantly decreased the level of plasma TG level to 18.11%, and increased the plasma TC and HDL-C level significantly to 58.96 and 76.43%, respectively, compared to the group treated by LPS. Also, administration of LPS-treated rats with the GANE (82 mg/kg.b.w.) significantly decreased the level of plasma TG level to 25.72%, and significantly increased the plasma TC and HDL-C level to 66.76 and 105.26% respectively, compared to the group treated by LPS. In addition, administration of LPS-treated rats with dexamethasone (0.5 mg/kg) significantly increased the level of plasma TG level to 5.51%, and significantly decreased the plasma TC and HDL-C level to 135.15 and 23.17% respectively, compared to the group treated by LPS.

Figure 6

Effect of GANE on levels of on plasma TC, TG and HDL (C) in control and treated rats.

Effect of GANEs on Oxidative Stress on the Lungs

Table shows lung superoxide dismutase (SOD), GPx, GSH, and MDA levels. Oral administration of LPS (200 μg/kg b.w.) led to decrease in lung SOD, GPx, and GSH significantly to 64.90, 66.11, and 56.93%, and significant decrease in the lung MDA to 171.17%, respectively, (P < 0.01) compared to the control group, which indicates acute lung fibrosis. Treatment of rats with the GANE (32.8 mg/kg.b.w.) significantly increased the level of the lung SOD, GPx, and GSH to 136.70, 87.10, and 83.28%, and led to a significant decrease in the lung MDA to 47.26%, respectively, compared to the group treated by LPS (P < 0.01). Also, administration of LPS-treated rats with the GANE (82 mg/kg.b.w.) significantly increased the level of lung SOD, GPx, and GSH to 160.45, 160.51, and 129.90%, and led to a significant decrease in the lung MDA to 99.99%, respectively, compared to the group treated by LPS (P < 0.01). While treatment of rats with dexamethasone (0.5 mg/kg) increased the level of lung SOD, GPx, and GSH significantly to 49.49, 56.52, and 36.98%, and led to a significant decrease in lung MDA to 32.49%, respectively, compared to the group treated by LPS (P < 0.01).

Table 3

Effect of GANEs on Levels of Lung SOD, Glutathione Peroxidase (GPx), Reduced Glutathione (GSH), and Malondialdehyde (MDA) in Control and Treated Ratsa

groups	treatment description	SOD (U/mg protein)	GPx (μmol of GSH oxidized/mg protein)	GSH (μg/mg protein)	MDA (μmol/mg tissue)
I	Normal control	210.74 ± 16.46	18.99 ± 3.5	7.22 ± 0.61	4.25 ± 0.46
II	LPS (200 μg/kg)	73.85 ± 7.63@	6.28 ± 1.33@	3.11 ± 0.38@	11.47 ± 2.80@
III	GANE (32.8 mg/kg) + LPS (200 μg/kg)	174.87 ± 12.47@	11.47 ± 1.86@	5.70 ± 0.5 @	6.07 ± 0.94@
IV	GANE (82 mg/kg) + LPS (200 μg/kg)	192.76 ± 16.10 @	16.81 ± 2.91@	7.15 ± 0.53 @	3.82 ± 0.67@
V	Dexamethasone (0.5 mg/kg) + LPS (200 μg/kg.)	110.71 ± 10.98@	9.83 ± 1.7@	4.26 ± 0.36@	7.05 ± 0.45@

Data shown are mean ± SD of number of observations within each treatment. Values are statistically significant at @P < 0.01. LPS (200 μg/kg.) treated rats were compared with normal control rats. Experimental groups (III–V) were compared with LPS treated rats.

Effect of GANEs on Inflammation Markers Levels in Lungs

Table reveals a significant increase (P < 0.01) in TNF-α and IL-6 in rats treated with LPS (200 μg/kg b.w.) to 360.78 and 331.74%, and a decrease in lung IL-4 (P < 0.01) to 47.85%, respectively, compared to the control. The administration of GANEs (32.8 mg/kg.b.w.) clarified a decrease (P < 0.01) in lung TNF-α and IL-6 in rats treated with LPS to 59.74 and 54.15%, and a significant increase in lung IL-4 (P < 0.01) to 47.52%, respectively, compared to LPS-treated groups. Administration of GANEs (82 mg/kg.b.w.) presents a decrease (P < 0.01) in lung TNF-α and IL-6 in rats treated with LPS to 69.08 and 65.64%, and a significant increase (P < 0.01) in lung IL-4 to 75.11%, respectively, compared to LPS-treated groups (P < 0.01). Also, administration of dexamethasone (0.5 mg/kg) found a significant decrease (P < 0.01) in lung TNF-α and IL-6 in rats treated with LPS to 72.76 and 73.50%, and significant increase in lung IL-4 (P < 0.01) to 83.11%, respectively, compared to LPS-treated groups (P < 0.01).

Table 4

Effect of GANEs on Levels of Lung TNF-α, Interleukin-4 (IL-4), and Interleukin-6 (IL-6) in Control and Treated Ratsa

groups	treatment description	TNF-α (pg/g tissue)	IL-4 (pg/g tissue)	IL-6 (pg/g tissue)
I	normal control	21.65 ± 4.00	75.82 ± 6.07	42.56 ± 5.24
V	LPS (200 μg/kg)	95.43 ± 3.91@	39.54 ± 5.10@	183.75 ± 14.66@
III	GANE (32.8 mg/kg) + LPS (200 μg/kg)	38.42 ± 2.49@	58.33 ± 4.59@	84.25 ± 3.30@
IV	GANE (82 mg/kg.) + LPS (200 μg/kg)	29.50 ± 4.53@	69.24 ± 6.64@	63.14 ± 6.02@
V	Dexamethasone (0.5 mg/kg) + LPS (200 μg/kg.)	25.99 ± 3.08@	72.40 ± 7.21@	48.76 ± 3.91@

Data shown are mean ± SD of number of observations within each treatment. Values are statistically significant at @P < 0.01. LPS (200 μg/kg) treated rats were compared with normal control rats. Experimental groups (III–V) were compared with LPS treated rats.

Figures –9 declare a significant (P < 0.05) increased in lung p38MAPK, TGF-β, and NF-κB genes expression to 354.74, 514.56, and 276.47%, respectively, in LPS-treated groups compared with the normal rats, expressing a severe lung damage. When rats were administered with GANEs at 32.8 mg/kg, they exhibited a significant decrease in lung p38MAPK, TGF-β, and NF-κB genes expression to 28.00, 59.24, and 56.77%, respectively, compared to rats treated with LPS. The administration of rats with GANEs at 82 mg/kg.b.w. declared a significant decrease in lung p38MAPK, TGF-β, and NF-κB genes expression to 59.49, 69.51, and 67.44%, respectively, compared to rats treated with LPS. Also, treatment of rats with dexamethasone (0.5 mg/kg) decreased the level of lung p38MAPK, TGF-β, and NF-κB genes expression significantly (P < 0.05) to 51.38, 56.87, and 47.65%, respectively, compared to rats treated with LPS.

Figure 7

Effect of GANEs (32.8 and 82 mg/kg.b.w.) on levels of lung p38MAPK gene expression in LPS-treated rats. Representative bar diagram of three independent experiments is presented.

Figure 9

Effect of GANEs (32.8 and 82 mg/kg.b.w.) on levels of lung NF-κB gene expression in LPS-treated rats. Representative bar diagram of three independent experiments is presented.

Effect of GANEs (32.8 and 82 mg/kg.b.w.) on levels of lung TGF-β gene expression in LPS-treated rats. Representative bar diagram of three independent experiments is presented.

Histopathological Examination

Histopathological examination of control lung tissue groups (I) showed within normal arrangement and appearance of the lung tissue with no fibrosis or inflammation × 200 H&E (Figure a). Moreover, the lung tissue of LPS-treated group (II), showed histological investigation with focal lesion consisted of thickening of alveolar walls with mild edema and few leukocytic cells infiltration (m and * respectively) X200H&E (Figure b). Also, the lung tissue showed alveolar lumen and alveolar wall recovery (#) in LPS-treated rats with administrated GANEs (32.8 mg/kg.b.w.) compared to the LPS-treated(Figure c) group (III). Furthermore, the histological examination of hepatocytes of groups (IV and V) showed a normal arrangement and apparency of the lung tissue with no fibrosis or inflammation (*) in LPS-treated rats administrated with GANEs (82 mg/kg.b.w.) and dexamethasone (0.5 mg/kg) compared to the LPS-treated group (Figure d,e) groups (IV and V).

Figure 10

Sections stained with hematoxylin and eosin (H&E; 400 X) for histological examination of lung tissues of different groups compared to the control group; (a) group I: Normal control; (b) group II: was administrated with LPS (200 μg/kg); (c) group III: was administrated with GANEs (32.8 mg/kg) + LPS (200 μg/kg); (d) group IV: was administrated with GANEs(82 mg/kg) + LPS (200 μg/kg); (e) group V: was administrated with dexamethasone (0.5 mg/kg) + LPS (200 μg/kg).

Discussion

In the current study, a new GANE formula was designed for anticancer and lung protective estimation on the LPS-induced lung damage in rats.

Nanotechnology has changed the treatment ways of cancer and is fundamentally changing the pattern of treatment. It has had a major impact in selectively identifying cancer cells, delivering drugs, and overcoming the limitations of chemotherapies.

Niosomes, which have a structure of bilayer and consisted of nonionic surfactants and cholesterol self-association in an aqueous phase, are one of the most promising drug carriers. Niosomes are nonimmunogenic, biodegradable, and biocompatible. They have a long shelf-life, are extremely stable, and allow for controlled and/or sustained medicines to be delivered on the target site.[45] In the present study, we used niosomes as a GA carrier. Further studies reported the ability of niosomes to entrap a wide range of drugs.[46−48]

Polyphenols can prevent cancer initiation and promotion through various mechanisms including inhibition of the activation of oncogenes and genes involved in oxidative stress and inflammation.[49,50] Polyphenols can protect against carcinogenesis by modulating epigenetic aberrations such as histone modifications, DNA methylations, and microRNAs.[51] GA is one of the known effective polyphenols for treatment different types of cancers such as breast, melanoma, pancreatic, and colon cancer.[52,53]

In this work, our results referred to GANE has cytotoxicity to A549 lung carcinoma cell line with a low IC50 value. This is consistent with former reports that revealed that GA has a cytotoxic effect on cancer cells.[54−59]

LPS is a known compound that induces ALI/ARDS and induces ALI by primarily dysfunctional pulmonary surfactants,[60] As host receptor(s) recognizes LPS first, it initiates activation of a number of signal transduction cascades in lung cells.

In this study, the administration of LPS significantly increased the TC and TG levels and decreased significantly the plasma HDL-C level, which is an indicator of lipid peroxidation. Further, oral administration of GANEs at 32.8 and 82 mg/kg.b.w. and dexamethasone (0.5 mg/kg) provided significant protection against the LPS- induced lung damage.

SOD, GPx, and GSH play an important biological function in antioxidant and cell protection processes by eliminating ROS to restrain cell damage.[61−63] The present results point to GANE-mediated induction of SOD, GPx, and GSH, which may helped in inhibition of the inflammatory response by reducing oxidative stress induced in LPS-treated rats. GANEs may protect cellular compounds from LPS oxidative damage via multiple mechanisms.[64] These include ROS scavenging, metal ion chelation, and antioxidant enzyme up-regulation and activation.[65,66] In the current study, GANEs act as antioxidants on two levels: removing ROS and inhibiting ROS formation. GANEs remove ROS at the first level through direct scavenging or modulation of antioxidant enzyme activity; while at the second level, they prevent ROS formation and inhibition of ROS producing enzymes.[66] Whereas, LPS induced the generation of ROS[67] and can cause a significant increase in lung enzymes and lipid peroxidation, and a significant increase in MDA and a drop-in antioxidant enzyme activity in lungs.[68]

In the present study, GANEs and dexamethasone induced transcription of IL-4, and suppresses TNF-α and IL-6 led to reduce ROS production and attenuate inflammation in vivo. For example, suppression of TGF-β led to inhibit the expression of p38MAPK and TGF-β gene expression.[69] On the other hand, inhibition of TNF-α and p38MAPK in GANE-treated rats led to decrease of IL-6 levels.[70] Also, TNF-α, IL-6, p38MAPK, TGF-β, and NF-κB reduced lung inflammation and ROS generation and inhibits adhesion molecule expression and monocyte adhesion in the lung tissue;[71−76] this effect appears to be mediated by antioxidant enzymes.[77] Our study hypothesized that IL-4 inhibits IL-6 and TNF-α and p38MAPK, TGF-β, and NF-κB gene expression, thereby preventing the degradation of lung tissue, protecting cell membrane integrity, and delaying the inflammation.

This study was confirmed by Wang et al. and Bataller et al., who reported that elevation of TNF-α and IL-6 levels in rats treated with LPS.[78,79] The GANE could exert its anti-inflammatory activity through certain mechanisms of (a) antioxidant and radical scavenging activities and (b) modulation of arachidonic acid metabolism (by regulating cyclooxygenase, phospholipase A2, and lipoxygenase) and nitric oxide synthase activity.[80] According to histopathological investigations, the GANE can protect lungs against LPS-induced lung fibrosis. This is evidenced by a reduced inflammatory response.

Overproduction of MDA and inflammatory mediators (TNF-α and IL-6) may cause DNA breakage. Although GANE treatment may enhance the repair of damaged DNA, it may also be a good protectors for lung tissues. High SOD, GPx activity in GANE-treated rats resulting in suppressed MDA production. Resynthesis of GSH also promotes DNA repair leading to lung protection.[81] In our study, we also demonstrate that GANE treatment reduced the increase in lung fibrosis in the lung from LPS-treated rats.

Furthermore, GANE proved efficacious to significantly lower total and biologically active lung TGF-β1 and NF-κB gene expression. TGF-β plays a central role in fibrotic disorders in different organs, including fibrosis of the lung. In fact, it stimulates collagen and fibronectin production in fibroblasts.[82] On the other hand, it can suppress the production of proteases that degrade the extracellular matrix.[83] TGF-β has been shown to be increased in LPS-induced lung fibrosis in the alveolar inflammatory infiltrate.[84]

The present finding regarding the immediate induction of TNF-α is compatible with previous observations,[85] and the present findings clearly demonstrate that induction of TNF-α by intravenous administration of bleomycin was mediated by activation of p38 MAPK. Moreover, we observed significant suppression of p38 MAPK expression in the LPS-induced lung fibrosis model. Some previous studies demonstrated that another GANE reduced lipopolysaccharide-stimulated secretion of p38 MAPK expression in rats.[86−88]

The protective effect of GANE against LPS-induced pulmonary fibrosis has not been reported according to our knowledge, and this may be the first study of its kind.

In the present study, GANE administration markedly suppressed apoptosis of the lung tissues. The role of p38 MAPK, TGF-β1, and NF-κB gene expression on apoptosis of various cell types is controversial, but it is possible that the suppression of apoptosis by inhibiting death signals.[89]

Conclusions

The study resulted in the observation that the GANE has a strong protective activity against LPS-induced pulmonary fibrosis through adjust the levels of biomarkers of oxidative stress and inflammation-mediated gene expression. Also, our investigation demonstrated that GANEs prevent the liberation of ROS for the damaged lung cell by inhibits lung IL-6 and TNF-α and p38MAPK, TGF-β, and NF-κB gene expression. This property leads us to imagine the existence of relation between GANE and cytokines and oxidative stress biomarkers, leading to a modulation of inflammatory process associated with lung fibrosis. It is clear that we will require further and detailed studies.

Experimental Section

Materials and Methods

Ethanol was obtained from Fisher Scientific UK, and phosphate buffer tablets (PBS) pH 7.4 was purchased from Bio Shop Canada Inc. Tween 80, DMN and cholesterol were obtained from Sigma Aldrich, USA.

Equipment and Material

Sonicator (Daihan, Korea) was used to form small vesicles niosomes and a cooling centrifuge (VS-18000M, Korea) was used to precipitate them. The UV absorbance of gallic acid was determined employing a spectrophotometer (JENWAY 6405, U.K.) at 280 nm. The UV spectra of free and niosomes encapsulated gallic acid were recorded by a UV spectrophotometer (Shimadzu, Japan).

In vitro drug release process was performed by Spectra/Por, MW cutoff12,000, Spectrum, Canada and a magnetic stirrer model (TK22, Kartell, Italy). The samples absorbance was determined at 280 nm by a spectrophotometer (UNICO UV-2000, China). The image of niosomes were investigated by TEM (JEOL JEM.1230, Japan). Moreover, the FTIR was conducted utilizing FT/IR-4100 (A Basic Vector, Germany).

Estimation of Cytokines

Lung cytokines: TNF-α, interleukin-6 (IL-6), and interleukin-4 (IL-4) were detected using a UV microplate reader (Thermo Electric Corp., Shanghai, China) by measuring the absorption at 450 nm.

Preparation of GANEs

Niosomes were formulated by a hydration method. Cholesterol and tween 80 (1:2) were dissolved in a round flask containing 50 mL of ethanol. Ethanol was evaporated by a rotary evaporator (63 °C, 50 rpm) to produce dry thin films. The formed thin film was hydrated by PBS (pH 7.4) containing 3 gm of gallic acid. The produced niosomes were displayed to sonication to form small vesicle niosomes. At the end, a cooling centrifuge was used to precipitate niosomes at (12,000 rpm for 20 min).[41]

Characterization of GANEs

Entrapment Efficiency

The supernatant having free GA was separated from the pellet (encapsulated one) by centrifugation (12,000 rpm, 30 min), the supernatant was isolated. The UV absorbance of gallic acid was determined at diverse concentrations at 280 nm. The calibration curve of gallic acid was obtained by drawing the absorbance against the concentration. The free gallic acid absorbance within the supernatant was specified spectrophotometrically at 280 nm.[39,40,90] The entrapment efficiency of niosomes was calculated from byCi: initial concentration, Cf: final concentration.

UV spectrophotometry of GANE

The UV spectra of free and niosomes encapsulated gallic acid were recorded by a UV spectrophotometer (Shimadzu, Japan).[91]

In Vitro Drug Release

The dialysis manner was used to find in vitro release for free and encapsulated GA in PBS (pH 7.4).[40] Two milliliters of niosomes-encapsulated GA were introduced in a dialysis bag of cellulose acetate and sunken in PBS (100 mL) with stirring magnetically at 60 rpm. One milliliter was possessed at fixed time periods (every 1 h) of the immersed solution, replacing it with an equal volume of freshly prepared PBS. The samples absorbance was determined at 280 nm. The experiment was ended when the concentration of GA in the surrounding medium is constant.

TEM

Niosome-encapsulated GA was stained negatively by a 1% of phosphotungstic acid and left to air-dry and the sample was incubated on the grid for 10 min approximately, and then analyzed.[92]

FTIR spectra were obtained on lyophilized free and encapsulated gallic acid. Samples were ground with KBr at a ratio of 1:100 , and then are compressed by a hydraulic press with a pressure of 15,000 lbs. The pellets of the samples were scanned over the spectrum range 400–4000 cm–1.

Determination of GANE Cytotoxicity on A549 Lung Carcinoma Cell Line

The GANE effect on the viability of A549cell lines was found by an assay of MTT. A 24-well plate contains 1.0 × 106 A549 cells/well, were exposed to GANE at concentrations of 31.5, 63.5, 125, 250, 500, and 1000 μg/mL for 24 h. A549 cells line without GANEs were used as control. Also, A549 cells line were cultured with MTT (20 μL/well of 5 mg/mL stock) about 4 h. The yellowish water-soluble MTT converts into formazan crystals water-insoluble which can be dissolved in 200 μL DMSO. A microplate reader was used to detect absorbance at 570 nm.

Animals

90 Albino rats of weigh 150 ± 7 g (60 for LD50 estimation and 30 for GANE anticancer efficiency) were brought from the animal house of National Cancer Institute, Cairo University, Giza, Egypt. The rats were provided water and a standard diet ad-libitum, observed daily, and kept in cages of polypropylene (20 cm × 34 cm x 47 cm) under a constant environmental condition throughout the experimental work. All experiments of animals were conducted according to the guidelines of Ethics Committee of the Faculty of Applied Health Sciences Technology, October 6 University, Egypt (registration no. 20210115).

LD50 Determination for GANE

The LD50 was determined in animal groups (each n = 10) administering GANE at doses of 800, 1200, 1600, 2000, 2400, and 2800 mg/kg orally. Saganuwan’s method[23] was used to determine the LD50 by the following formulaDm: is the maximum dose that kills all animals. Z: is the average of dead animals between two consecutive groups. d: is a factor between two consecutive doses. n: is the animals number in each group.

Animal Treatments

A 200 μg LPS was injected slowly intratracheal at day 1 and day 15 to induce Lung fibrosis in rats.[30] GANE was suspended in 1% Tween 80 and then administrated to rats by intragastric intubation. Table showed the different groups of normal and treated rats.

Table 5

Treated Animal Groups

group	name of the group	treatment description
I	control	3 mL distilled water, orally for 7 weeks
II	LPS	intratracheal injection of 200 μg LPS in 1% Tween 80, at day 1 and day 15 (30)
III	GANE + DMN	rats were treated with GANE 1/50 LD50 (32.8 mg/kg) suspended in 1% (Tween 80, orally) for 15days + 200 μg LPS in 1% Tween 80, at day 1 and day 15
IV	GANE + LPS	rats were treated with GANE 1/20 LD50 (82 mg/kg) suspended in 1% (Tween 80, orally) for 15days + 200 μg LPS in 1% Tween 80, atday 1 and day 15
V	Dexamethasone + LPS	rats was intraperitoneal injected with dexamethasone (0.5 mg/kg) suspended in 1% Tween 80 + 200 μg LPS in 1% Tween 80, at day 1 and day 15

On 16th day of the experiment, the blood samples were assembled in heparinized tubes, then centrifuged for 20 min at 1000×g. Blood samples were used to evaluate plasma levels of cholesterol, TGs, and cholesterol-high density lipoproteins. Commercial kits (Asan and Youngdong Pharmaceutical Co., Korea) were used for the analysis.

Lung Specimens

The lung tissue was homogenized in 3 mL of PBS (pH 7.5) and centrifuged (3000 g, 10 min). The supernatant was used to assess SOD, GSH, and TBARs calorimetrically using a kit of the Cayman Chemical Company (An Arbor, MI). The activity of GPx was measured indirectly by determination of NADPH oxidation at 340 nm.

Quantitative PCR Real-Time

The total RNA amount was extracted from lung of the rats, and parts of (10–15 μg) from the isolated RNA were exposed to PCR analysis in real time, by Sepasol-RNA1Super according to instructions of the manufacturer. Steps of RT-PCR gene expression have been measured. The expression level of a transforming growth factor-β (TGF-β1), P38 protein mitogen-activated kinase (p38MAPK), and nuclear factor-kappaB (NF-κB) was quantified. Tests were carried out in a 50 mL single-plex reaction mixture. The reaction was performed under conditions of pre-incubation at 50 °C for 2 min, followed by 10 min with 40 cycles of 95 °C in 15 s and 60 °C in 1 min, respectively. The sequences of the primers were illustrated in the following Table .

Table 6

Primers sequences Used in Real-Time PCR

gene	primer sequence
p38MAPK	F: 5′-AGGGCGATGTGACGTTT-3′ R: 5′-CTGGCAGGGTGAAGTTGG-3′
TGF-β1	F:5′-ATTCAAGTCAACTGTGGAG-3′ R: 5′-: CGAAAGCCCTGTATTCCGTCT-3′
NF-κB	F: 5′- -GCGAGAGGAGCACAGATACC-3′ R: 5′-GCACAGCATTCAGGTCGTAG-3′
GAPDH (internal control for qRT-PCR)	F: 5′-CTCAACTACATGGTCTACATGTTCCA-3′ R: 5′-CCATTCTCGGCCTTGA-CTGT-3′
β-actin (internal control for qRT-PCR)	F-5′-GATTACTGCTCTGGCTCCTGC-3′ R-5′-GACTCATCGTACTCCTGCTTGC-3

Histological Assessment

The lung tissues were subdivided into pieces for treatment by formalin (10% buffered formaldehyde solution). Samples were introduced in paraffin, sectioned, and stained with hematoxylin and eosin. The changes were observed by a light microscope.

Statistical Analysis

The obtained results were expressed as mean ± SD for six separate determinations for spectrophotometric and ELISA measurements as well as three separate determinations for the invitro cytotoxicity and PCR analysis of genes expression. All the data were analyzed by SPSS/20 Software using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. P < 0.01 were considered to indicate statistical significance.