Immunomodulatory effect of turmeric, amla, and pepper mixture against sepsis-induced acute lung injury in rats

doi:10.21203/rs.3.rs-1626104/v1

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Immunomodulatory effect of turmeric, amla, and pepper mixture against sepsis-induced acute lung injury in rats

https://doi.org/10.21203/rs.3.rs-1626104/v1

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Acute lung injury (ALI), is a severe inflammatory lung disease. We tested the prophylactic effect of a functional food comprising three different anti-inflammatory plant products: turmeric, amla, and black pepper (TAB) against lipopolysaccharide (LPS)-induced ALI in rats. Two-month-old male Wistar rats were randomly divided into three groups: control (C), LPS (5mg/kg), and LPS with TAB (TAB). After 6 hours of LPS injection, the rats were sacrificed by cervical decapitation to collect the lung tissue. Results showed that TAB partially ameliorated LPS-induced circulating inflammatory cytokines (TNFα and IL6) and significantly prevented lung histopathological changes. TAB also suppressed LPS-activated ER stress markers (GRP78, pIRE1, and CHOP) and apoptotic proteins (caspase-3 and -12) in the lung. The anti-inflammatory effects of the TAB support its potential use at least as an adjuvant to mitigate ALI. Importantly, the ingredients of TAB are in use for centuries as part of their diet with limited or no toxic effects identified.

Inflammation

cytokines

immunomodulation

herbal formulation

functional food

lipopolysaccharide

Acute lung injury (ALI), and its severe condition, acute respiratory distress syndrome (ARDS) are serious inflammatory lung diseases affecting around 1 million people annually resulting in high morbidity and mortality ^{1, 2}. The most common cause of ALI is sepsis, due to microbial infection including current SARS-CoV-2 ^{3, 4}. Increased vascular permeability, leukocyte recruitment, and overproduction of cytokines causing alveolar and interstitial pulmonary edema, alveolar collapse, and hypoxemia are the characteristics of ALI ⁵. Despite much research, no effective therapies are available for ALI / ARDS in clinical practice. As of now, corticosteroid therapy in ALI is beneficial only in the early phase of lung inflammation but with several adverse effects like hyperglycaemia, hypokalaemia, dyslipidaemia, hypertension, peptic ulcers, immunosuppression, neuropsychiatric disturbances, osteoporosis, myopathy, etc ⁶. Excessive recruitment of immune cells in the lungs contributes to pulmonary damage as they disturb cellular homeostasis ⁷. Scientific evidence exists on the role of an overactive inflammatory response in the pathogenesis of early steps of ALI / ARDS. The host’s uncontrolled inflammatory response inclusive of leukocyte recruitment and/or their activation could impair the host pulmonary epithelial or endothelial layer. Hence, a strong immunomodulatory/ anti-inflammatory agent or formulation could alleviate this pathology.

Turmeric (Curcuma longa), Amla (Emblica Officinalis Gaertn.), and Black pepper (Piper nigrum L.) are popular culinary plants and are also used for several centuries in the Indian Ayurvedic medicine system for curing numerous inflammatory disorders. Turmeric is a food component used in many parts of the world. Curcumin, responsible for the vibrant yellow color of turmeric and the major active principle of turmeric has been attributed to numerous pharmacological activities of which the best-explored action is its anti-inflammatory effects ⁸. Curcumin was shown to modulate the inflammatory response by suppressing the production of tumor necrosis factor-alpha (TNF-α), IL-1, -2, -6, -8, and − 12 ⁹. While one of the major limitations of curcumin is its low bioavailability black pepper was shown to increase curcumin’s bioavailability by 20 folds ^{10, 11}. Further, black pepper itself has potential anti-inflammatory activity and has been commonly utilized in food preparation and traditional medicine in several countries ^{12, 13}. Studies have also shown that amla extract possesses strong anti-inflammatory effects and inhibited microbial infection-induced expression of the neutrophil chemokines interleukin (IL)-8, GRO-alpha, GRO-gamma, and pro-inflammatory cytokine, IL-6 in the bronchial epithelial cells ¹⁴. The immunomodulatory effect of amla is also evident ¹⁵. The synergistic effects of turmeric, amla, and pepper are well evident in the scientific literature ^16–19. Earlier we reported the prophylactic efficacy of these three components individually against diabetic complications under in vitro conditions and in animal models ^20–23 which motivated us to investigate the combined effect of turmeric, amla, and black pepper (TAB) against sepsis-induced ALI in a rat model.

2.1. Chemicals and reagents:

Lipopolysaccharides from Escherichia coli O55:B5 (LPS), horseradish peroxidase (HRP) conjugated anti-rabbit, and anti-mouse antibodies, immunoblot chemicals, and protease inhibitor cocktail were purchased from Sigma Chemicals (MO, USA). Primary antibodies for NF-kB, pNF-kB, PI3K, AKT, pAKT, ATG5, LC3, BAX, caspase 3, and caspase 12 were purchased from Cell Signaling Technology, (Massachusetts, USA). TNFa, pERK1, pJNK1, pPI3K, GRP78, pIRE1, CHOP, Beclin1, P62, Bcl2 and β-actin antibodies were purchased from Thermo Scientific (Massachusetts, USA).

2.2. Animal Experiment:

Two-month-old Wistar rats were received from the animal facility of ICMR- National Institute of Nutrition and adapted for a week. Rats were fed with an AIN93 pellet diet and drinking water ad libitum. The animal room was maintained at standard conditions of 22 ± 2^oC temperature, 50–60% of humidity, and 12:12 h dark: light cycle. All the procedures of animal work were approved and accorded to the Institutional Animal Ethical Committee. Rats were then randomly divided into the following three groups: control (C), LPS, and LPS with a functional food mixture of turmeric, amla, and black pepper (TAB). The group-TAB was pre-treated with the TAB in the diet for a week

2.3. Functional food mixture of turmeric, amla and black pepper (TAB):

Fine quality dried turmeric root/rhizome and black pepper (dried fruits) were purchased from a local market and grounded to a fine powder. Good quality fresh amla was collected in the winter season from a local market and washed under tap water, air-dried to remove water, seeds were removed, cut into small pieces and air-dried at room temperature under shade. The dried amla pieces were grounded into a fine powder and stored in an airtight glass jar. One g of amla powder, 0.25 g of turmeric powder, and 0.5 g of black pepper were added to 100 g of the AIN93 rodent powder diet and mixed thoroughly. These specified individual doses were decided based on the active principle content (curcumin in turmeric, β-glucogallin in amla, and piplartine in black pepper) required for optimal efficacy, back-calculated based on our earlier studies using in vitro and in vivo models against diabetic complications ^20–27.

LPS was administered on the eighth day to all rats except group-C at a dose of 5 mg/kg body weight, dissolved in saline via intraperitoneal. The LPS dose used in the study was based on our pilot study and publications of others ^{28, 29}. After 30, 60, and 120 min of LPS injection blood sample was collected from the retro-orbital plexus for analysis of inflammatory cytokines. Rats were sacrificed by cervical decapitation after 6 hours of LPS injection and a part of lung tissue was fixed in formalin solution and the remaining was snap-frozen for further analysis.

2.4. Analysis of circulatory cytokines by ELISA:

Plasma concentrations of cytokines IL-6, IL-10 and TNF-α were measured by the Solid Phase Sandwich ELISA method (R&D Systems, MN, USA) by following the manufacturer's instructions. Briefly, 100 uL of plasma sample was mixed with 100 µL of the Detection Antibody, diluted in 1% BSA in PBS, pH 7.2 and incubated for 2 hours in dark at room temperature. After washing with 0.05% Tween 20 in PBS, pH 7.2, added 100 µL of the working dilution of Streptavidin-HRP conjugate, covered the plate and incubated for 20 minutes at room temperature. Following washing, add 100 µL of Substrate Solution (1:1 mixture of H2O2 and Tetramethylbenzidine) and incubated at room temperature for 20 min. 50 µL of 2 N H2SO4 was added to each well to stop the reaction and gently tapped the plate to ensure proper mixing. Optical density at 450 nm was measured immediately, using a microplate reader. A standard curve in the range of 62.5 to 4000 pg/ml was constructed by reducing the data generating a four-parameter logistic (4-PL) curve-fit.

2.5. Immunoblotting:

Lung tissue was homogenised in 20 mM Tris lysis buffer (100 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 µg/ml each of aprotinin, leupeptin, and pepstatin; pH7.5) and protein concentration was measured by the Lowry method. An equal amount of protein from each of the three experimental groups was subjected to SDS PAGE and the resolved bands were transferred to nitrocellulose membrane to probe with primary antibodies each at a specific dilution. Then incubated with respective secondary antibody conjugated with HRP enzyme. Finally, the bands are visualised after developing with enhanced chemiluminescence detection reagent (Bio-Rad Laboratories, California, USA) and images were captured by an Image Analyzer (G-Box iChemi XR; Syngene, Maryland, USA). Images were quantitated and analyzed using ImageJ software (NIH, Bethesda, USA).

2.6. Immunohistochemistry:

The lung tissue was perforated with and fixed in formalin solution and embedded in paraffin. The lung sections (4 µm thickness) were stained with hematoxylin and eosin (H&E) and were visualized in a microscope (Leica Microsystems, Germany) at 10X and 40X magnifications.

2.7. Statistical analysis:

Results are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the one-way ANOVA by GraphPad Prism 8 scientific software (GraphPad Software, California, USA). Tukey's multiple comparisons test was used for post hoc analysis. p < 0.05 was recognized as statistically significant.

3.1. TAB ameliorated LPS-induced inflammatory markers:

We observed no changes in the food intake and body weights among the experimental groups (Fig. 1A & B).

Tumour Necrosis Factor-alpha (TNFα), an inflammatory cytokine secreted by macrophages during acute inflammation, triggers several cell-signalling events. We have observed higher circulating TNFα by 30 min of LPS injection and reached peak by 60 min and later diminished by 120 min but still higher than control. Pre-treatment with TAB could partially ameliorate the rise in TNFα levels as seen in Fig. 1C. Another important inflammatory cytokine, Interleukin 6 (IL6) responsible for stimulating acute phase inflammatory response was increased in circulation by 60 min and went on increasing till 120 min of LPS injection. However, TAB could completely ameliorate the rise of IL6 levels at 60 min and partially at 120 min of LPS injection (Fig. 1D). Interleukin 10 (IL-10), a potent anti-inflammatory cytokine that plays a vital role in avoiding damage to the host and preserving tissue homeostasis was found increased by 30 min of LPS injection and gradually decreased but greater than control even at 120 min. TAB intervened rats showed moderate levels of circulatory IL10 throughout the observed period (Fig. 1E). Immunoblotting of cytokines at the tissue level showed higher TNFα levels in the lungs of LPS injected rats while TAB intervention significantly prevented this rise of TNFα levels (Fig. 1F&G). Nuclear factor kappa-B (NF-κB) is a "rapid-acting" primary transcription factor activated by TNFα, IL6, IL1β and others, plays a central role in the regulation of inflammation through its ability to induce transcription of proinflammatory genes including cytokines, chemokines, and adhesion molecules ^{30, 31}. We observed increased phospho-NF-κB expression in the lungs of LPS injected rats while TAB pre-treatment significantly prevented LPS induced NF-κB activation (Fig. 1F&G).

3.2. TAB prevented LPS-induced lung histopathological changes:

Inflammatory infiltrates, alveolar membrane thickening and severe alveolar space destruction were observed in LPS injected group compared to the control group (Fig. 2). However, pre-treatment with the TAB significantly prevented the severity of ALI compared with the rats that received LPS alone (Fig. 2).

3.3. TAB ameliorated LPS-induced MAPK (ERK/JNK) and PI3K/Akt pathway activation:

There are several signalling pathways associated with the inflammation, however, MAPKs and PI3K/Akt are considered as two key signalling cascades affecting the translocation of NF-κB during inflammation ³².

MAPKs are well-known signalling adaptors involved in inflammatory responses. To determine the outcome of TAB pre-treatment on LPS-induced MAPKs, the levels of extracellular signal-regulated protein kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) were assessed by immunoblot. We observed increased levels of pERK1/2 and JNK1 protein expression in the lungs of the LPS group that were prevented by TAB (Fig. 3).

Phosphoinositide 3-kinases (PI3Ks) regulate inflammatory response by modulating the activation and spreading of neutrophils and macrophages ³³. Hence, we next investigated the status of PI3K and Akt protein expression. We observed increased phosphorylation of PI3K and Akt proteins in the LPS group that were prevented by TAB (Fig. 3).

3.4. TAB ameliorated LPS-induced ER stress:

ER stress response plays a vital role in many infectious conditions including LPS induced sepsis and hence, we next investigated the status of ER stress markers in the lung of LPS induced inflammatory group and the TAB pre-treatment group. As shown in Fig. 4, we observed increased protein expression of ER stress markers like GRP78, pIRE1 and CHOP in the LPS group. However, TAB pre-treatment prevented LPS-induced ER stress.

ER stress is a protective response not only to restore protein homeostasis but also to restore cellular homeostasis by modulating several cellular signalling pathways by activating the unfolded protein response. ER stress recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway and is a major contributor to several inflammatory diseases ^{34, 35}. The ER stress inhibitor 4-PBA was shown to prevent LPS induced inflammation through modulating ER stress and autophagy in ALI models ^{36, 37}. In the current study, the TAB could alleviate ER stress in LPS injected rats (Fig. 4). The tannoids of amla, curcumin and piperine present in the TAB were earlier shown to combat ER stress individually by various mechanisms in varied disease models ^38–40.

3.5. TAB ameliorated LPS-induced changes in Autophagy- lysosomal system:

In investigating possible mechanisms of TAB interference in reducing inflammation, we analysed the level of Beclin1, ATG5, LC3-II and p62 proteins to inspect the possible alterations in the autophagy-lysosome system (Fig. 5). LPS-group showed increased protein expression of Beclin1, ATG5, LC3II and P62 while TAB- group showed a beneficial effect in preventing these alterations (Fig. 5).

3.6. TAB ameliorated LPS-induced Apoptotic markers:

We next investigated the status of apoptotic protein expression in the lungs of experimental rats. As shown in Fig. 6, the LPS- group showed increased protein levels of Bax that plays a crucial role in the mitochondrial apoptotic process. Bax/Bcl2 ratio estimated by quantitative immunoblotting was greater in LPS-group when compared with the control group but with no statistical significance. Caspase-3 protein another marker of apoptosis was elevated in LPS-group but prevented in TAB-group. Cleaved caspase 12, a marker of ER stress-induced apoptosis was elevated in LPS-group which is partly prevented in TAB-group.

Turmeric (Curcuma longa L.) is commonly used as a spice and also in Ayurvedic medicine for curing inflammatory conditions. It comprises three curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin), volatile oils (natlantone, tumerone and zingiberone), proteins, sugars and resins with an excellent safety profile ⁴¹. Turmeric offers several biological activities most of which are attributed to the presence of curcumin. Curcumin exerts strong anti-oxidant and anti-inflammatory activities and more than 100 clinical trials were conducted on the role of curcumin in various diseases including autoimmune disorders ⁴². Curcumin was shown to regulate the inflammatory response by down-regulating the activity of cyclooxygenase-2 (COX-2), lipoxygenase, and inducible nitric oxide synthase (iNOS) enzymes. It also inhibits the production of the inflammatory cytokines TNF-α, IL-1, -2, -6, -8, and − 12, and down-regulates mitogen-activated JNK ^{9, 43, 44}. Curcumin dose-dependently inhibited LPS-induced NF-κB with an IC50 of 18 µM, interestingly turmeric extract showed an IC50 of 15 µM ⁴⁵. In another study, curcumin-free turmeric was also shown to have anti-inflammatory activity ⁴⁶.

Amla plant has been used in treating several disorders especially inflammatory diseases such as pneumonia, hepatitis, and even cancer, and is well-known for a wide range of biological activities ⁴⁷. Phytochemical studies on amla showed that it is rich in tannins, polyphenols, flavonoids, gallic acid, vitamin C, and emblicol ⁴⁸. The immunomodulatory effects of Amla were mainly due to the down-regulation of pro-inflammatory genes, COX-2, iNOS, IL-16, IL-6, and TNF-α ^{49, 50}. Amla also alleviated LPS induced inflammation in macrophages by decreasing the release of pro-inflammatory mediators ⁵¹. Chang et al. reported that beta-glucogallin isolated from amla could inhibit LPS-induced oxidative stress by preventing activation of JNK and p38 in murine macrophages ⁵².

Black pepper is used in traditional food formulations, perfumery, alternative medicine, and cosmetics in many Asian countries. The active phenolic component of pepper, piperine, is shown to have beneficial health effects. Piperine was shown to inhibit the translocation of activator protein 1 (AP-1) in IL1β-treated cells thereby modulating inflammation ⁵³. Piperine was reported to exert anti-inflammatory activity by decreasing the expression of ICAM-1 on the macrophage surface thereby inhibiting macrophage polarization ⁵⁴. Piperine could suppress the pro-inflammatory factors IL-1β and TNF-α, and enhance the anti-inflammatory effects of IL-10 and TGF-β1 ⁵⁵. Though curcumin has several health benefits, one of the drawbacks is its poor bioavailability. Interestingly, piperine was found to increase the bioavailability of curcumin by 2000% in both humans and animals with no adverse effects ¹⁰. Hence, the issue of curcumin’s poor bioavailability could be resolved by the addition of pepper, consequently enhancing the efficacy of the formulation by synergism.

In conclusion, pre-treatment with the TAB constituting turmeric, amla and black pepper powder at specified doses prevented LPS induced ALI in rats. TAB has ameliorated LPS-induced inflammation and lung tissue damage by modulating ER stress response, MAPK signalling, autophagy and cell death processes. The anti-inflammatory effect of the TAB is well supported by these results and hence, can be used at least as an adjuvant for ALI and other inflammatory conditions. The formulation can be easily prepared as the ingredients are economical and readily available. Further, people regularly consume these constituents for centuries as part of the diet without any signs of toxicity.

Funding:

Author G.B.R has received grants (Q-11/16/2019-R&D) from Ministry of Food Processing Industries, Government of India.

Competing Interests:

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions:

MN: Performing the experiments, data collection and analysis

KKK, KPR, MS and URA: data collection and analysis

GBR: Project administration, manuscript review and funding acquisition

SSR: Study conception and design, supervision and manuscript writing

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Immunomodulatory effect of turmeric, amla, and pepper mixture against sepsis-induced acute lung injury in rats

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Chemicals and reagents:

2.2. Animal Experiment:

2.3. Functional food mixture of turmeric, amla and black pepper (TAB):

2.4. Analysis of circulatory cytokines by ELISA:

2.5. Immunoblotting:

2.6. Immunohistochemistry:

2.7. Statistical analysis:

3. Results And Discussion

3.1. TAB ameliorated LPS-induced inflammatory markers:

3.2. TAB prevented LPS-induced lung histopathological changes:

3.3. TAB ameliorated LPS-induced MAPK (ERK/JNK) and PI3K/Akt pathway activation:

3.4. TAB ameliorated LPS-induced ER stress:

3.5. TAB ameliorated LPS-induced changes in Autophagy- lysosomal system:

3.6. TAB ameliorated LPS-induced Apoptotic markers:

4. Conclusions

Declarations

References

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