Amelioration of autophagy and inflammatory signaling pathways via α-lipoic acid, burdock and bee pollen versus lipopolysaccharide-induced insulin resistance in murine model

Lipopolysaccharide (LPS) has previously been implicated in insulin resistance by generating an innate immune response and activating inflammatory cascades. Many studies have discovered a relationship between high levels of serum LPS and the advancement of diabetic microvascular problems, indicating that LPS may play a role in the control of critical signaling pathways connected to insulin resistance. The current study focused on signaling pathways linked to insulin resistance and explored probable mechanisms of LPS-induced insulin resistance in a murine model. It next looked at the effects of burdock, bee pollen, and -lipoic acid on LPS-induced inflammation and autoimmune defects in rats. LPS intoxication was induced via ip injection for one week in a dose of 10 mg/kg followed by α-lipoic acid, Burdock and bee pollen in an oral treatment for one month. Following that, biochemical and molecular studies were performed. The RNA expression of the regulating genes STAT5A and PTEN was measured. In addition, ATF-4 and CHOP as autophagy biomarkers were also subjected to mRNA quantification. The results demonstrated a considerable improvement in the -lipoic acid, Burdock, and bee pollen treated groups via modifying oxidative stress indicators as well as molecular ones. Furthermore, glucose concentration in serum and α-amylase were also improved upon treatment with the superiority of α-lipoic acid for modulating all estimated parameters. In conclusion: the results declared in the current study suggested that α-lipoic acid could regulate insulin resistance signaling pathways induced by LPS intoxication.


Introduction
Diabetes mellitus is a chronic metabolic disorder defined by an excessively high level of blood glucose. Insulin deficiency, either absolute or relative, induced by insulin resistance or pancreatic β-cell malfunction are major causes of hyperglycemia [1]. The incidence of diabetes mellitus injured almost 460 million individuals globally and anticipated to reach to 700 million by 2045 [2], which pose a serious risk to world health. The basic pathophysiological processes of diabetes include hyperglycemia, dyslipidemia, insulin resistance, and metabolic pathway dysregulation, which contribute different problems involving various organ functions [1]. With a better knowledge of diabetes, oxidative stress, immunological abnormalities, and genetic mutations, are now recognized as major Germany). Primers for PTEN, STAT-5A, ATF-4 and CHOP gene expression were purchased from thermos Fisher Co. (Waltham, AM, USA). Kits for antioxidant determination were obtained from Biodiagnostic Co. (Giza, Egypt). All other chemicals were of the greatest analytical grade.

Animals
In the current study, 48male Wister albino of 200 gm ± 20 were obtained from animal house, National Research Center (NRC). Animals were kept in room temperature and under ordinary light/dark cycle. Furthermore, they were kept in suitable cages (8/each) and freely provided by water pelleted food ration. All animals were kept in the above mentioned conditions for one week prior to procedure starting point for acclimatization. All animals in the current experiment were approved by The Animal Care and Use Committee of NRC's ethical standards and guidelines (24411122022).

Experimental design
To start the experiment, rats were divided into six groups as described below: Group 1: Negative control healthy animals that were administered saline. Group 2: Lipopolysaccharide intoxicated rats through ip injection for one week (total dose 20 mg/kg) and served untreated [33].
Group 4: Rats treated post lipopolysaccharide intoxication via an oral dose of bee pollen (3 mg/kg daily) for one month [35]. Group 5: Lipopolysaccharide-intoxicated rats then were treated by Burdock in a dose of 5 mg/kg daily for one month [36].
Group 6: Injected lipopolysaccharide animals then given a one-month oral treatment regimen consisting of a mixture of α-lipoic acid, bee pollen and burdock.

Blood sampling
At the end of the experiment, animals were subjected to anesthesia using Co 2 . Blood samples were collected from the retro-orbital vein. Additionally, all sera were separated post centrifugation.

Determination of serum malondialdehyde
Using assays that are available for purchase and were given by Biodiagnostic Company, malondialdehyde reactivity was assessed (Giza, Egypt) [37].

Glutathione reductase
Glutathione reductase activity was estimated using biodiagnostic kits (Giza, Egypt) [38]. Quantitative Real Time Polymerase Chain Reaction for gene expression. Total RNA was isolated from blood samples using the Qiamp small kit as directed by the manufacturer (Qiagen; USA). Next, using one-step QuantiTecht SYBR green (Qiagen; USA) kits, RT-PCR analysis was done to determine the mRNA gene expression of PTEN, STAT-5A, ATF-4, and CHOP. Forward and reverse primer sequences were presented in Table 1. Denaturing double-stranded cDNA was used for the quantification process, which next involved annealing genomic DNA to forward and reverse primers at temperatures appropriate for each primer. To assess each gene's relative expression against -actin as a reference gene, a comparative CT (2-ΔΔCT) method was applied [39].

NMR spectroscopy
The NMR spectra of lipopolysaccharide were recorded on a Jeol 500-MHz (Japan) NMR spectrometer equipped. Samples were dissolved in DMSO. Signals were referenced to internal acetone (H 2.225 ppm, C 30.89 ppm), and the standard of software was used to acquire and process these data [40].

Gene
Forward sequence Reverse sequence

Statistical analysis
The acquired information was examined using SPSS software, version 16. The mean SE was used to express each value. Following a one-way analysis of variance, Turkey's multiple comparisons post hoc test was employed to look at statistically significant differences between groups (ANOVA). When P value less than 0.05, significance was taken into account.

Oxidative stress modulation
Malondialdehyde levels significantly increased in the lipopolysaccharide-impaired groups, reaching 88.9 nmol/ml compared to the negative control group (13.866 nmol/ml). However, glutathione levels significantly decreased, falling to 0.547 mmol/g of tissue from 3.103 mmol/g in healthy animals. Malondialdehyde levels were also shown to be clearly modulated across all treatment regimens, particularly with -Lipoic acid, indicating the most substantial influence (Figs. 1 and 2).

Estimation of glucose level and α-amylase in serum
Lipopolysaccharide intoxication recorded a significant elevation in glucose level recording 169.16 mg/dl as compared to negative control animals (91 mg/dl). Meanwhile, a considerable fall in glucose level was explored after -lipoic acid to be close to normal value (93.1212 mg/dl) demonstrated -lipoic acid's superiority in controlling glucose level in serum sample. Nevertheless, bee pollen therapy and combined treatment groups show no change in glucose levels. Nonetheless, a considerable improvement in glucose level was observed after burdock therapy, but it was smaller than that reported in the -lipoic acid treated group. (Table 2).
On the other hand, α-amylase concentration recorded a significant reduction (149.79 U/L) as comparing to healthy groups (542.66 U/L). Additionally, -lipoic acid therapy results in a considerable improvement of 444.266 U/L. (Table 3).

Modulation of PTEN and STAT-5A gene expression
As compared to the negative control animal, lipopolysaccharide intoxication increased PTEN and STAT-5A gene expression by 1.7 and 5 fold changes, respectively. Meanwhile, a significant reduction in PTEN as well as STAT-5A was investigated upon α-lipoic acid to be close to normal value (1 and 0.8 fold changes) reflecting the superiority of α-lipoic acid in modulating PTEN and STAT-5A gene expression. Treatment with bee pollen, on the other hand, shows no improvement in PTEN gene expression. Nonetheless, a considerable modification in STAT-5A gene expression after bee pollen administration was found to be around normal value.
Burdock treatment as well as combination of all treatment regimens notably declared downregulation in the expression of PTEN and STAT-5A genes (Figs. 3 and 4).

Regulation of autophagy signaling pathway
Data in the current study declared a significant reduction in ATF-4 gene expression post lipopolysaccharide intoxication recording 0.479 fold changes as compared to healthy group. On the other, a significant elevation was recorded in the expression of CHOP gene with 4.9 fold change as compared to negative control group. All of the above-mentioned regimens resulted in an apparent modification of ATF-4 gene expression near normal levels, with the superiority of the bee pollen-treated group recoding a 0.96 fold change. Additionally, a substantial downregulation was seen following treatment with -lipoic acid, bee pollen, Burdocks, and a combination of all treated groups, with the superiority of α-lipoic acid treatment group recording 0.8 fold changes as compared to negative control group. (Figs. 5 and 6).

NMR spectroscopy of LPS
Complex organic solvent solutions, such DMSO, were able to produce well-resolved NMR signals when LPS, which still contains the hydrophobic lipid A moiety, was exposed to NMR analysis. The inner core oligosaccharide and other parts, such lipid A, are frequently thought to contribute to the endotoxicity of LPS, which is usually believed to be the endotoxic moiety of the entire LPS molecule (Figs. 7-9).

Discussion
Insulin resistance is the most common risk factor for metabolic syndrome. As it reduces the response of target cells to insulin owing to a decline in their sensitivity to insulin secretion, insulin resistance has a negative effect on the overall metabolism [41]. Characteristic outcomes caused by insulin resistance include metabolic disorders, such as type 2 diabetes, obesity and glucose intolerance. Insulin resistance is also recognized as a cardiovascular disease related factor and is characterized by endothelial dysfunction [42].
On the other hand, LPS from gut microbiota was reported as linker between inflammation and insulin resistance-induced metabolic syndrome [10]. However, whether LPS directly regulated signaling pathways associated with insulin resistance, the related molecular mechanisms were less studied. Herein we reported that LPS elevated oxidative stress biomarkers as well as mRNA gene expression of   both STAT-A5 and PTEN in addition to a notably increase of autophagy signaling pathways ATF-4 and CHOP. It was reported that the LPS could possibly induce oxidative stress in mice as well in rats due to the tissue damage which increased the production of pro-inflammatory cytokines and generation of ROS. The capability of LPS for induction of oxidative stress was previously studied in murine model [43].
One of the various effects of oxidative stress is peroxidation of unsaturated fatty acids in membrane. The degree of lipid peroxidation, measured as MDA, was used to explain the role of LPS in peroxidation of lipids. The concentration of MDA was increased   immediately upon LPS administration [44]. Different previous studies investigated significant elevation of MDA levels upon LPS intoxication [45]. Furthermore, lipid peroxidation levels were elevated upon high doses of LPS in Fischer rats [46]. Also, LPS (200 μg/mouse, for 2 h) induced lipid peroxidation increment [47]. In the current study, animals treated byα-lipoic acid only as well as combination treated group declared a significant modulation of oxidative stress. Previously, it was documented that α-lipoic acid plays an important role in modulating the adverse effect of LPS-induced oxidative stress that may develop multiple organ failure [48,49].
Furthermore, as previously recorded, diabetes is considered as an autoimmune disease as well as metabolic one. 3. Among diabetic subjects, suggesting that LPS induced inflammatory responses could accelerate different metabolic disorders including diabetes. Data recorded form the current study declared a significant elevation of serum blood glucose level upon LPS intoxication.
Consistent with our findings, previous study investigated that macrophages may respond to LPS challenge in a more exaggerated manner under hyperglycemic conditions than under normal glucose conditions, and noticed hyperglycemia via activation of signal transduction and subsequent cytokine production in LPS stimulated monocytic cells [50].
Our study declared a significant reduction in glucose level upon α-lipoic acid treatment. Improvement of glucose levels by α-lipoic acid has been previously recorded in T2DM cases through improving the diabetes-related deficit in glucose metabolism in addition to activation of insulin signaling pathways [51]. Furthermore, their finding declared the possible correlation between oxidative stress  induced by LPS and insulin resistance [52].
On the other hand, burdock treatment as well as bee pollen doesn't declare any improvement in glucose. These results are in contrast to the previous in vitro study investigation which proved the efficacy of burdock in lowering glucose concentration [53].
Herein, a significant reduction was noticed in the expression of PTEN upon LPS administration. Furthermore, a significant improvement was declared upon α-lipoic acid, burdock as well as combination treatment. On the other hand, Bee pollen didn't declare remarkable modulation in PTEN gene expression. Conversely, studies have reported that polymorphisms in PTEN are associated with increased insulin resistance or metabolic syndrome, a major risk for the onset of T2D. For example, substitution of cytosine to guanine in position 9 in the 50-untranslated region in PTEN is commonly found in Japanese individuals with T2D. Indeed, individuals with this polymorphism have increased PTEN protein levels, decreased AKT phosphorylation, and increased insulin resistance when compared with healthy controls [54]. Furthermore, metabolic syndrome individuals declared a reduction in PTEN promoter supporting that PTEN down-regulation has a protective effect in T2D [55]. The PTEN knockout (KO) mice models have been the most useful experimental tools in elucidating the metabolic activities of PTEN in vivo. Mice with PTEN loss only in metabolic tissues, as opposed to mice with whole-body PTEN KO, are alive and enable for the examination of the tissue-specific contributions of PTEN signaling in affecting insulin action and glucose homeostasis [55]. The most of mice with fibroblast KO for PTEN in metabolic organs recorded improvement in metabolic functions, including greater glucose tolerance and insulin sensitivity [56,57].
PTEN as a tumor suppressor is involved in the proliferation of various cells [58]. The reduction of PTEN expression leads to the activation of the PI3K/Akt signaling pathway [59]. Previous study recorded that LPS-induced inhibition of PTEN is directly related to fibroblast proliferation, differentiation and collagen secretion by modulating PI3K/Akt/GSK3β pathway. Furthermore, PTEN expression was previously reduced in vitro upon LPS intoxication; the underlying mechanism may be correlated with LPS-induced activation of some transcription factors as NF-κB and c-Jun [60].
The current finding declared a significant downregulation in groups treated by burdock as well as combination group. This modulation may be caused by a burdock active ingredient that has numerous pharmacological properties and has been shown to have anti-tumor, neuroprotective, antiviral, anti-inflammatory, anti-oxidant, and endoplasmic reticulum (ER) stress regulating actions through regulating NFkB, PI3K/AKT, and Stat pathways [61][62][63].
Furthermore, a remarkable overexpression in STAT5A gene was demonstrated upon LPS intoxication. Meanwhile an obvious downregulation was declared upon treatment with all treated regimens (α-Lipoic acid, Bee pollen and Burdock). Previous findings suggested that STAT5A participating in suppression of cytokine signaling pathways via STAT gene expression regulation [64].
Autophagy signaling pathways of both ATF-4 and CHOP recorded a significant reduction in their expression upon LPS intoxication. Consistent with our findings, it was demonstrated that hyperglycemia triggered ATF-CHOP signaling pathways upon LPS intoxication that it may promote pro-inflammatory response in macrophages [65].
Furthermore, NMR spectroscopy analysis elucidated the 4-phosphate that is sterically nearby can be replaced by the negatively charged carboxyl group, which is next to the lipid A backbone. In prior research, interactions of intact LPS with natural pathogenrelated receptors (CD14, MD-2 and TLR4) in almost natural settings were examined. Conformational characteristics of these interactions revealed knowledge of different biological roles, including insulin resistance.

Conclusion
In conclusion, the current study confirmed that LPS intoxication is associated to dysregulation of both inflammatory and autophagy signaling pathways which leading to insulin resistance and in hence causing a notable elevation in glucose blood level. The impact of α-Lipoic acid, Bee pollen and Burdock on modulation of all dysregulated biochemical parameters was investigated in the current study. In addition, data in our findings declared the superiority of α-Lipoic acid in modulating all dysregulated parameters. Therefore, it could be concluded that the administration of α-Lipoic acid may be a promising regimen for modulating oxidative stress as well as insulin resistance induced by LPS intoxication [40].

Author contribution statement
Rehab Abdel-Megeed: Conceived and designed the experiments; Performed the experiments; Wrote the paper. Mai O. Kadry: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Data availability statement
Data included in article/supplementary material/referenced in article.