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.