{"paper_id":"ca5f96a3-fba5-4e40-8cfa-3b00ddd1ccfc","body_text":"Andrographolide ( 1 , Fig.  1 ) is the active component of the medicinal plant  Andrographis paniculata  Nees (Acanthaceae) 1 . A.  paniculata  Nees is traditionally used in China, India, and Thailand in removing heat and toxic materials 1 . Andrographolide has been employed to treat inflammation- and oxidative stress-related diseases, including diarrhea 2 , rheumatoid arthritis 3 , and chronic rhinosinusitis with nasal polyps 4 . Andrographolide treatment reduces serum cholesterol, triglycerides, and low-density lipoprotein cholesterol in hyper-cholesterolemic patients and animals fed with high-fat diets 5 . Andrographolide treatment decreases hepatic neutrophil/macrophage infiltration, down regulates local inflammation, and reduces liver damage in thioacetamide-induced mouse hepatic fibrosis 6 . The anti-inflammatory effect of andrographolide is induced by inhibiting the NF-κB signaling pathway 7 – 9 . Mass spectrometry result and molecular docking analysis revealed that andrographolide binds to the NF-κB p50 subunit at Cys62 position 10 . Andrographolide has potent inhibitory effect to the NF-κB signaling pathway in several disease models, including TNBS-induced colitis mouse model 11 , lipopolysaccharide (LPS)-induced acute lung injury 9 , and endometriosis 12 . Figure 1 Synthesis of andrographolide derivatives.\nSynthesis of andrographolide derivatives.\nSeveral andrographolide derivatives have been synthesized to improve the potency and selectivity of drugs. The andrographolide derivatives isoandrographolide, 7- O -methylwogonin, and skullcapflavone-I significantly inhibit the release of NO and prostaglandin E2 and the production of interleukin (IL)-1β and IL-6 in LPS-stimulated J774A.1 macrophage cells in a dose dependent manner 13 . AL-1, an andrographolide analog, improves insulin resistance by down regulating the NF-κB signaling pathway 5 . Treatment with 40 mg/kg and 80 mg / kg AL-1exhibits a significant hypoglycemic effect by reducing cholesterol levels and increasing high density lipoprotein levels, respectively 5 . At 1 μM, AL-1 suppresses the high-glucose-induced phosphorylation of the NF-κB p65 subunit and IκBα in rat-islet-derived insulinoma cells. In this case, homeostatic assessment shows that insulin resistance was reduced, and insulin sensitivity was restored 5 . Andrographolide suppresses toll-like receptor 4 (TLR4) expression and NF-κB signaling in multiple myeloma cells at 10 μM 14 . However, the suppression of TLR4 and NF-κB possibly resulted from andrographolide cytotoxicity against the cell model under conditions specified by the experiment 14 .\nTLRs are involved in the regulation of innate and adaptive immunity, and non-infectious inflammatory liver diseases 15 . For example, TLR4 acts as a receptor for LPS, which is a cell-wall component of Gram-negative bacteria that induces strong inflammation 15 . TLR4 is closely related to lung infection 15  and hepatocellular carcinoma 16 . Intracellular TLRs sense foreign genomic material: TLR3 detects viral double-stranded RNA 17 , TLR7 recognizes single-stranded RNA upon viral infection 18 , and TLR9 responds to specific unmethylated CpG motifs in microbial genomic DNA 19 . TLR hyperactivation by corresponding ligands generates a cytokine storm that ultimately mediates acute tissue injury 20 – 22 . The MyD88/NF-κB/JNK complex is an important collection of adaptor molecules that links receptor-initiated signals to transcriptional regulation in the nucleus 21 . Innate immune responses to several TLRs triggered by ligands also share this highly conserved signaling pathway. Therefore, targeting the NF-κB signaling pathway may elucidate TLR-initiated innate immunity, such as acute pulmonary injury 23 .\nIn the current study, we synthesized a set of andrographolide derivatives (Fig.  1 ) to explore new conceptual compounds with higher rigidity, which possibly restricts the rotation of the bonds of C9-C11 and C11-C12 and lowers the competence as Michael acceptor 24 . We then determined the specificity of these andrographolide derivatives against NF-κB-dependent and -independent TLR signaling pathways. One major function of andrographolide is to treat respiratory infection and inflammation 25 . We also evaluated the protective effectiveness of active andrographolide derivative on LPS-induced acute pulmonary inflammation. We aimed to determine the structural basis for the improved potency and immune-suppressive effect of andrographolide. We proposed the structure-activity relationship of andrographolide derivatives against NF-κB signaling pathway, and determined the therapeutic potential in LPS-triggered acute pulmonary injury.\n\nCompounds of listed andrographolide derivatives were synthesized as shown in Fig.  1  and  S1  (Supporting information). First, to obtain the aromatic derivatives  2a  or  2b , 4-nitro-2-methoxyphenol was introduced into the 14-position of andrographolide ( 1 ) or its 14-epimer ( 12 ) via the Mitsunobu reaction in a process modified from our previous report 26 . The 3,19-diol derivatives  3a  and  3b  resulted from deprotecting 3, 19-acetonylidene from  2a  and  2b . Anhydrous ZnCl 2  catalyzed the di-acetylation of  3a  and  3b  to yield  4a  and  4b , respectively. Meanwhile, the triethylamine-catalyzed mono-acetylation of  3a  and  3b  formed the 19-acetylated products  5a  and  5b , respectively. The 3-alcohols of  5a  and  5b  were oxidized by Dess-Martin periodinane into the corresponding 3-ketones,  6a  and  6b , respectively. Reducing the lactone moiety of  2b  yielded a semi-reduction product of semi-acetal  7b  and a fully reduced product of alcohol  8b . Deprotection of 3,19-acetonylidene from  7b  and  8b  yielded  9b  and  10b , respectively. Semi-acetal  7b  was transformed into the diastereomeric acetals of  11ba  and  11bb  (Figure  S1 ), which was inseparable by routine separation methods. Diastereoisomeric separation of  11ba  and  11bb  was performed through the 3,19-disilylated modification of  11ba  and  11bb  to form separable  12ba  and  12bb , respectively, followed by the deprotection of 3,19-disilylation from  12ba  and  12bb , respectively (Figure  S1 ).\nWe utilized luciferase reporters bearing the TNF-α/NF-κB, TLR4/NF-κB, and interferon-γ activated signal (GAS, Table  1 ) to examine the activity of the newly synthesized andrographolide analogs on signaling pathways that govern the inflammatory response. Inhibiting TLR4 signaling has received considerable attention in the regulation of innate immune response. LPS-activated TLR4 regulates the expression and translocation of NF-κB 27 . Interferon-γ (IFN-γ) helps in activating anti-microbial and anti-tumor lymphocytes to regulate the proliferation and differentiation of biomolecules 28 . The inhibition of IFN-γ signaling regulates cytokine expression through the IFN-γ-STAT3-GAS signaling pathway. These derivatives did not show detectable cytotoxicity against both AD-293 and HeLa cell lines. Parent compound  1  did not show any influence against these signaling pathways up to 10 μM in AD-293 and HeLa cells (Table  1 ). Meanwhile, none of these derivatives influenced active against the IFN-γ signaling pathway. The 14α-compounds of  2a  and  3a  inhibited TLR4/NF-κB, but not TNF-α/NF-κB in both AD-293 and HeLa cell lines. The 14β-compound  2b  influenced TLR4 and only affected the NF-κB in HeLa cells. 14β-Compound  3b  showed a similar effect on TLR4 to  2b ; however, but the inhibitory effect of the former against NF-κB improved to lower than 10 μM in both cell lines. The latter result indicates that removing 3,19-acetonylidene facilitates the NF-κB inhibition from  2b  to  3b . Di-acetylation of  3a  and  3b  generated diverse results of the corresponding  4a  and  4b  products. In particular,  4a  selectively exerted its activity against NF-κB in the AD-293 cell line; however,  4b  influenced NF-κB only in the HeLa cell line. Furthermore, results indicated that  4a  was the most active compound against TLR4, followed by  4b . Unlike the di-acetylation substances, mono-acetylated  5a  and  5b  exhibited good activity against TLR4 and NF-κB in the two cell lines.  5a  and  5b  suppressed TLR4 to similar levels; however,  5b  had slightly more active inhibitory effect against NF-κB than that of  5a . The oxidization of 3-alcohol  5a  into ketone  6a  enhanced the inhibition of TLR4 expression and suppression of NF-κB signaling in the AD-293 cell line. Oxidation also removed the inhibitory effect on NF-κB in the HeLa cell line. Compound  6b  did not active influence NF-κB in both cell lines and had reduced effect on TLR4 relative to 3-alcohol  5b . The semi-acetal  7b  only affected the NF-κB in the HeLa cell line. Unfortunately, modifying compounds  8b ,  9b ,  10b ,  11ba , and  11bb  completely reduced lactone, thereby removing their inhibitory effect on NF-κB and TLR4. All of the lactone-modified derivatives of  7b ,  8b ,  9b ,  10b ,  11ba , and  11bb  analogs were inactive in all of the tests, although all of the other derivatives potently inhibited the TLR4 signaling pathway in the AD-293 cells (Table  1 ). Therefore, this work identified the lactone moiety as the key structural motif in the interaction between the test compound and its drug target(s). Notably, the 14α-isomers of  4a  and  6a  influenced NF-κB signal transduction more in the AD-293 cell line than in the HeLa cell line. The 14β-isomers of  2b ,  4b , and  7b  also selectively inhibited the NF-κB signaling pathway in the HeLa cell line. Overall, among all of the tested derivatives, the NF-κB signaling pathway in the AD-293 cells was only potently inhibited by the 4-h treatment of  3b ,  5b , and  5a  at EC 50  concentrations of approximately 10 μM in AD-293 cells (Table  1 ). These derivatives were also sensitive to the NF-κB signaling pathway in HeLa cells (Table  1 ), thereby suggesting that the inhibitory activities of these compounds are unaffected by the cellular background. Table 1 The EC 50  (half effective concentration) of all compounds against NF-κB, TLR4, and IFN-γ signaling pathways and the cytotoxicity (CC 50 , half cytotoxic concentration) in AD-293 and Hela cells. cmpd EC 50  in AD-293 (μM) EC 50  in Hela (μM) NF-κB a TLR4 b IFN-γ c CC 50 d NF-κB a CC 50 d Andrographolide ( 1 ) >10 >10 >10 >10 >10 >10 \n 2a \n >10 5.76 ± 1.40 >10 >10 >10 >10 \n 2b \n >10 3.35 ± 0.36 >10 >10 6.22 ± 0.60 >10 \n 3a \n >10 5.36 ± 0.58 >10 >10 >10 >10 \n 3b \n 8.97 ± 1.03 3.49 ± 0.34 >10 >10 2.00 ± 0.66 >10 \n 4a \n 8.85 ± 1.06 0.90 ± 0.14 >10 >10 >10 >10 \n 4b \n >10 3.32 ± 0.72 >10 >10 5.48 ± 0.39 >10 \n 5a \n 10.37 ± 0.71 5.79 ± 0.96 >10 >10 4.89 ± 0.28 >10 \n 5b \n 7.54 ± 1.33 5.98 ± 0.30 >10 >10 2.31 ± 0.60 >10 \n 6a \n 7.92 ± 0.42 3.55 ± 0.07 >10 >10 >10 >10 \n 6b \n >10 7.67 ± 0.67 >10 >10 >10 >10 \n 7b \n >10 >10 >10 >10 5.48 ± 0.34 >10 \n 8b \n >10 >10 >10 >10 >10 >10 \n 9b \n >10 >10 >10 >10 >10 >10 \n 10b \n >10 >10 >10 >10 >10 >10 \n 11ba \n >10 >10 >10 >10 >10 >10 \n 11bb \n >10 >10 >10 >10 >10 >10 \n 12ba \n >10 >10 >10 >10 >10 >10 \n 12bb \n >10 >10 >10 >10 >10 >10 a Treated for 4 h. b Treated for 16 h. c Treated for 24 h. d Treated for 24 h.\nThe EC 50  (half effective concentration) of all compounds against NF-κB, TLR4, and IFN-γ signaling pathways and the cytotoxicity (CC 50 , half cytotoxic concentration) in AD-293 and Hela cells.\na Treated for 4 h.\nb Treated for 16 h.\nc Treated for 24 h.\nd Treated for 24 h.\nWe then utilized the TLR4 ligand LPS to induce inflammatory responses in THP-1 cells. The LPS-stimulated transcription of pro-inflammatory cytokine IL-6 was suppressed with  3b  and  5a  treatments at 5 and 10 μM (Fig.  2A ), respectively. However, IL-18 transcription was unchanged (Fig.  2B ). Treatment with  3b  and  5a  abrogated the transcription of polyinosinic:polycytidylic acid (poly I:C)-induced IL-6 (Fig.  2C ). Upon poly I:C stimulation,  5a  treatment enhanced IL-18 mRNA, which remained unchanged with  3b  treatment (Fig.  2D ). This result was consistent with a previous report that IL-18 transcription was unrelated to TLR stimulation 29 . Treatment with  3a ,  5b , and  6b  also inhibited LPS or poly I:C-stimulated transcription of IL-6 (Fig.  2E,F ). Figure 2 Andrographolide derivatives inhibited proinflammatory cytokine transcription. THP-1 cells differentiated with 10 −8  M PMA were treated with 1 μg/ml LPS in the presence or absence of  3b  and  5a  for 6 h. The mRNA levels of ( A ) IL-6 and ( B ) IL-18 were determined by real-time PCR analysis. THP-1 cells were transfected with 50 ng/μl poly I:C using PEI in the presence or absence of  3b  and  5a  for 9 h. The mRNA levels of ( C ) IL-6 and ( D ) IL-18 were determined by real-time PCR analysis. ( E ) THP-1 cells were treated with 5 μg/ml LPS in the presence or absence of 10 μM  3a ,  5b , and  6b  for 6 h, or ( F ) THP-1 cells were transfected with 50 ng/μl poly I:C in the presence or absence of 10 μM  3a ,  5b , and  6b  for 9 h, the mRNA level of IL-6 was determined by real-time PCR analysis. Results are presented as mean ± SD from at least three independent experiments (** p  < 0.05;  n.s ., non-specific; −, DMSO control).\nAndrographolide derivatives inhibited proinflammatory cytokine transcription. THP-1 cells differentiated with 10 −8  M PMA were treated with 1 μg/ml LPS in the presence or absence of  3b  and  5a  for 6 h. The mRNA levels of ( A ) IL-6 and ( B ) IL-18 were determined by real-time PCR analysis. THP-1 cells were transfected with 50 ng/μl poly I:C using PEI in the presence or absence of  3b  and  5a  for 9 h. The mRNA levels of ( C ) IL-6 and ( D ) IL-18 were determined by real-time PCR analysis. ( E ) THP-1 cells were treated with 5 μg/ml LPS in the presence or absence of 10 μM  3a ,  5b , and  6b  for 6 h, or ( F ) THP-1 cells were transfected with 50 ng/μl poly I:C in the presence or absence of 10 μM  3a ,  5b , and  6b  for 9 h, the mRNA level of IL-6 was determined by real-time PCR analysis. Results are presented as mean ± SD from at least three independent experiments (** p  < 0.05;  n.s ., non-specific; −, DMSO control).\nTo better understand the effect of andrographolide derivatives on TLR-triggered innate immune responses, we further analyzed the cytokines released into the medium under different stimuli. Treatment with  3b  and  5a  significantly inhibited the LPS-stimulated release of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6 (Fig.  3A–C ), as well as poly I:C-triggered TNF-α expression (Fig.  3D ). Treatment with  3a ,  5b , and  6b  suppressed the LPS-stimulated expression of IL-1β, TNF-α, and IL-6, respectively (Fig.  3E–G ). However, their inhibitory effect against TNF-α and IL-6 expression (Fig.  3F ) was less than that of  3b  and  5a  (Fig.  3B,C ). Compounds  3a ,  5b , and  6b  inhibited poly I:C-triggered TNF-α under the same conditions (Fig.  3H ). Compounds  3a  and  5b  expressed more inhibitory activity against poly I:C-induced TNF-α than compounds  3b ,  5a , and  6b  (Fig.  3D–H ). The NF-κB signaling pathway was indirectly inhibited by  3a  and  6b  (Table  1 ). These compounds exerted their inhibitory activity through TLR4 signal transduction. Figure 3 Andrographolide analogs inhibited pro-inflammatory cytokines expression. THP-1 cells differentiated with 10 −8  M PMA were treated with 1 μg/ml LPS in the presence or absence of  3b  and  5a  for 6 h. The levels of secreted ( A ) IL-1β, ( B ) TNF-α, and ( C ) IL-6 in the medium was determined by LEGENDplex TM  bead-based immunoassays. ( D ) THP-1 cells were transfected with 50 ng/μl poly I:C in the presence or absence of  3b  and  5a  for 9 h. The level of secreted TNF-α in the medium was determined as in ( B ). Effects of  3a ,  5b , and  6b  on the LPS-stimulated secretion of ( E ) IL-1β, ( F ) TNF-α, and ( G ) IL-6 were determined as in ( A ). ( H ) Effects of  3a ,  5b , and  6b  on the poly I:C-stimulated secretion of TNF-α were determined as in ( A ). Results are presented as mean ± SD of three different experiments (** p  < 0.05; −, DMSO control).\nAndrographolide analogs inhibited pro-inflammatory cytokines expression. THP-1 cells differentiated with 10 −8  M PMA were treated with 1 μg/ml LPS in the presence or absence of  3b  and  5a  for 6 h. The levels of secreted ( A ) IL-1β, ( B ) TNF-α, and ( C ) IL-6 in the medium was determined by LEGENDplex TM  bead-based immunoassays. ( D ) THP-1 cells were transfected with 50 ng/μl poly I:C in the presence or absence of  3b  and  5a  for 9 h. The level of secreted TNF-α in the medium was determined as in ( B ). Effects of  3a ,  5b , and  6b  on the LPS-stimulated secretion of ( E ) IL-1β, ( F ) TNF-α, and ( G ) IL-6 were determined as in ( A ). ( H ) Effects of  3a ,  5b , and  6b  on the poly I:C-stimulated secretion of TNF-α were determined as in ( A ). Results are presented as mean ± SD of three different experiments (** p  < 0.05; −, DMSO control).\nIn several diseases, the activation of the NF-κB signaling pathway governs the transcription of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α 30 . NF-κB activation depends on the phosphorylation-triggered proteasome degradation of inhibitor IκB proteins. The IκB kinase complex, which is responsible for IκB phosphorylation, is essential to the nuclear translocation of NF-κB and the initiation of subsequent signal transduction 30 . To confirm the inhibitory activity of andrographolide derivatives against NF-κB signaling, we determined the effect of these analogs on the nuclear translocation of NF-κB in AD-293 cells using immunofluorescent staining. Treatment with  3b  and  5a  inhibited the TNF-α-stimulated nuclear translocation of p65 at 10 μM (Fig.  4A ). By contrast, structural analogs  3a ,  5b , and  6b  did not show apparent activity at 10 μM (Fig.  4B ). Western blot analysis revealed that all the compounds decreased LPS-triggered phosphorylation of p65 and IκBα in THP-1 cells (Fig.  4C ). Compounds  3b  and  3a  enhanced the expression level of p65 as compared with LPS alone (Fig.  4C ). Compound  6b  inhibited the endogenous expression level of p65 subunit (Fig.  4C ). LPS-stimulated expression of IκBα was affected by none of the compounds (Fig.  4C ). Figure 4 Treatment with andrographolide analogs inhibited the NF-κB signaling pathway. AD-293 cells were treated with 50 ng/ml TNF-α alone or in combination with either ( A )  3b  and  5a , or ( B )  3a ,  5b , and  6b  for 3 h. After fixation, the nuclear translocation of NF-κB p65 was examined under a confocal microscope. Images were obtained under 200× magnificence. ( C ) Western blot analysis of the levels of phosphorylated p65, p65, phosphorylated IκBα, and IκBα treated with 1 µg/ml LPS alone or in combination with compounds  3b ,  5a, 3a ,  5b , and  6b  in THP-1 cells. The blot of actin was used as an internal loading control. The results are representative of three independent experiments.\nTreatment with andrographolide analogs inhibited the NF-κB signaling pathway. AD-293 cells were treated with 50 ng/ml TNF-α alone or in combination with either ( A )  3b  and  5a , or ( B )  3a ,  5b , and  6b  for 3 h. After fixation, the nuclear translocation of NF-κB p65 was examined under a confocal microscope. Images were obtained under 200× magnificence. ( C ) Western blot analysis of the levels of phosphorylated p65, p65, phosphorylated IκBα, and IκBα treated with 1 µg/ml LPS alone or in combination with compounds  3b ,  5a, 3a ,  5b , and  6b  in THP-1 cells. The blot of actin was used as an internal loading control. The results are representative of three independent experiments.\nProteasome inhibitor MG 132 abrogates the activation of the NF-κB signaling pathway by blocking IκB degradation 31 . In Fig.  5 , the LPS-triggered expression of IL-1β and TNF-α slightly increased in the presence of MG132 compared with LPS treatment alone. This condition may result from the activation of other signaling mediators, such as activator protein-1, which controls the transcription of pro-inflammatory cytokines 31 . Pretreatment with MG132 significantly decreased the suppressive effect of the andrographolide derivatives  3b ,  5a ,  3a ,  5b , and  6b  on LPS-stimulated IL-1β and TNF-α production (Fig.  5A,B ). The strongest counteractions were exerted by  3a  against IL-1β production and by  5b  against TNF-α production. Thus,  3a  and MG132 directly competed for IL-1β production, whereas  5b  and MG132 competed directly for TNF-α production. Synthetic CpG oligonucleotides (ODNs) contain unmethylated CpG dinucleotides and are present in bacterial DNA at a higher frequency than in mammalian DNA. Class A CpG ODN 2216 induces the TLR9-dependent activation of pro-inflammatory cytokines by weakly stimulating NF-κB signaling 30 . Treatment with compounds  3b ,  5a , and  3a  did not inhibit ODN 2216-induced TNF-α and IFN-β transcription (Fig.  6 ). By contrast, treatment with  5b  and  6b  strongly enhanced the transcription of TNF-α and IFN-β cytokines, respectively (Fig.  6C,D ). These results show a similar effect on LPS-stimulated TNF-α cytokine levels (Figs  3F,H  and  5B ). Figure 5 Effects of proteasome inhibitor MG-132 on the inhibition of TLR4 signaling induced by andrographolide derivatives. THP-1 cells differentiated with 10 −8  M PMA were treated with 1 μg/ml LPS and pretreated with 1 μg/ml MG-132 16 h before the addition of andrographolide analogs. The levels of ( A ) IL-1β and ( B ) TNF-α in the medium were determined by LEGENDplex TM  bead-based immunoassays after treatment with compounds  3b ,  5a ,  3a ,  5b , and  6b  for an additional 6 h. The results are representative of at least three independent experiments and presented as mean ± SD of three different experiments (** p  < 0.05; −, DMSO control). Figure 6 Effects of andrographolide derivatives on class A CpG ODN2216-stimulated TLR9 signaling. AD-293 cells were stimulated with ODN2216 alone or combined with andrographolide derivatives. The mRNA levels of TNF-α and IFN-β treated with ( A ) and ( B )  3b  and  5a , or ( C ) and ( D )  3a ,  5b , and  6b , respectively, were quantitated by real-time PCR. The results are representative of at least three independent experiments and presented as mean ± SD (** p  < 0.05;  n.s ., nonspecific).\nEffects of proteasome inhibitor MG-132 on the inhibition of TLR4 signaling induced by andrographolide derivatives. THP-1 cells differentiated with 10 −8  M PMA were treated with 1 μg/ml LPS and pretreated with 1 μg/ml MG-132 16 h before the addition of andrographolide analogs. The levels of ( A ) IL-1β and ( B ) TNF-α in the medium were determined by LEGENDplex TM  bead-based immunoassays after treatment with compounds  3b ,  5a ,  3a ,  5b , and  6b  for an additional 6 h. The results are representative of at least three independent experiments and presented as mean ± SD of three different experiments (** p  < 0.05; −, DMSO control).\nEffects of andrographolide derivatives on class A CpG ODN2216-stimulated TLR9 signaling. AD-293 cells were stimulated with ODN2216 alone or combined with andrographolide derivatives. The mRNA levels of TNF-α and IFN-β treated with ( A ) and ( B )  3b  and  5a , or ( C ) and ( D )  3a ,  5b , and  6b , respectively, were quantitated by real-time PCR. The results are representative of at least three independent experiments and presented as mean ± SD (** p  < 0.05;  n.s ., nonspecific).\nWe further evaluated the therapeutic potential of active andrographolide derivative on LPS-induced acute pulmonary injury in mice (Fig.  7 ). Oral administration of compound  3b  did not significantly change the body weight during the treatment (Fig.  7A ). Hematoxylin and eosin (H&E) staining results indicated that compound  3b  decreased LPS-induced alveolar wall thickening (upper panel, Fig.  7B ). Neutrophil infiltration is an indicator of lung injury 32 . We then determined neutrophil infiltration by immunohistochemistry staining of myeloperoxidase (MPO), which is most abundantly expressed in neutrophils 32 . The number of MPO-positive cells were significantly increased in the LPS-treated group, whereas compound  3b  decreased MPO-positive cells (lower panel, Fig.  7B ). Phosphorylated p65 was mainly localized in the nucleus in the LPS-treated group, whereas compound  3b  led to the cytosolic localization of phosphorylated p65 (Fig.  7C ). The expression level of phosphorylated p65 was undetectable by Western blot analysis (Fig.  4C ); however, fluorescent staining is not a quantitative method, and fluorescent intensity shown in different treatment could not reflect protein expression levels. LPS-induced elevation of serum levels of pro-inflammatory cytokines and chemokine, including IFN-β, TNF-α, and MCP-1, were significantly decreased by compound  3b  treatment (Fig.  7D–F ). These results suggested that compound  3b  protected LPS induced pulmonary injury through the inhibition of NF-κB signaling  in vivo . Figure 7 Efficacy of compound  3b  (45 mg/kg) on LPS-induced pulmonary injury. ( A ) Body weight changes of mice with LP-induced pulmonary injury. ( B ) H&E staining and immunohistochemistry analysis of MPO of mice lung tissue (630× magnification). ( C ) Immunofluorescent analysis of phosphorylated p65 level in mice lung tissue (400× magnification). Serum levels of ( D ) IFN-β, ( E ) TNF-α, and ( F ) MCP-1 were determined by LEGENDplex TM  bead-based cytokine and chemokine immunoassays. The results are representative of two independent experiments (n ≥ 4 for each group) and presented as mean ± SD ( p  value was labeled on the figure;  n.s ., nonspecific).\nEfficacy of compound  3b  (45 mg/kg) on LPS-induced pulmonary injury. ( A ) Body weight changes of mice with LP-induced pulmonary injury. ( B ) H&E staining and immunohistochemistry analysis of MPO of mice lung tissue (630× magnification). ( C ) Immunofluorescent analysis of phosphorylated p65 level in mice lung tissue (400× magnification). Serum levels of ( D ) IFN-β, ( E ) TNF-α, and ( F ) MCP-1 were determined by LEGENDplex TM  bead-based cytokine and chemokine immunoassays. The results are representative of two independent experiments (n ≥ 4 for each group) and presented as mean ± SD ( p  value was labeled on the figure;  n.s ., nonspecific).\n\nIn summary, we identified several andrographolide derivatives as specific NF-κB inhibitors (Table  1 ). SARs indicated that in addition to the importance of the good pharmacophore of 4-nitro-2-methoxyphenoxy, the derivative’s inhibitory potency and selectivity of a derivative are determined by 14-stereochemistry, 13-monoacetylation, 3,19-diacetylation, 3-alcohol, or 3-ketone. Among these andrographolide derivatives,  3b  is a potent and specific NF-κB inhibitor that prevents the phosphorylation of the NF-κB p65 subunit without affecting the endogenous expression of NF-κB family members. In this report, we identified compounds  3b ,  5a ,  5b , and  6b  as potential immunomodulatory inhibitors of TLR signaling with distinct regulation of NF-κB family members. Compound  3b  effectively reduced LPS-induced pulmonary injury by decreasing the nucleus translocation of phosphorylated p65 and serum pro-inflammatory cytokines. The andrographolide derivative  3b  may serve as a useful framework in further developing potential agents for the treatment of inflammatory and autoimmune diseases, and immunotherapy against various pathogens, such as bacteria-induced acute pulmonary injuries.\n\nCell culture media and fetal bovine serum (FBS) were purchased from Gibco. Lipopolysaccharide (LPS), polyinosinic:polycytiylic acid (poly I:C), and ODN 2216 were purchased from InvivoGen (San Diego, CA). TNFα was purchased from PeproTech (Rocky Hill, NJ). Chemical compounds were dissolved in DMSO at 10 mM as store solution. The content of DMSO doesn’t exceed 0.1% in cells. MG132 was purchased from MedChem Express (Monmouth Junction, NJ, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) except for those otherwise noted.\nCell lines were obtained from the American Type Culture Collection (ATCC). HeLa and AD-293 cells were maintained in DMED high glucose medium supplemented with 10% FBS. Hela and AD-293 stable cell lines harboring NF-κB and GAS response element in pGL4.20 vector (Promega) were maintained in the presence of 1 μg/ml puromycin. AD-293 cells with overexpression of TLR4 and NF-κB response element plasmid was maintained in the presence of 1 μg/ml puromycin and 10 μg/ml blasticidin. HEK-293 cells with overexpression of TLR9 were maintained in the presence of 10 μg/ml blasticidin.\nTHP-1 cells were maintained in RPMI-1640 medium with 10% FBS plus 0.05 M 2-mercaptoethanol. The differentiation of THP-1 monocytes into macrophages-like cells was triggered by 10 −8  M phorbol 12-myristate 13-acetate (PMA) for 72 hours. All cell lines were maintained in a humidified incubator with an atmosphere of 95% air and 5% CO 2  at 37 °C.\nReporter cells were treated with 50 ng/ml TNF-α to stimulate NF-κB signaling pathway, 20 pg/ml IFN-γ to stimulate GAS signaling pathway, and 1 μg/ml LPS to stimulate TLR 4. Medium was removed at the end of the treatment, and cell extracts were prepared and luciferase activity was measured by Luciferase assay kit (Promega) according to the manufacturer’s instructions. EC 50  was defined as the concentration of drug that inhibited stimulator-triggered luciferase reporter activation by 50% after continuous drug exposure for 4 hours.\nWestern blot was performed according to a standard protocol. The primary antibodies using in this research are from NFκB pathway sampler kit (CST#9936), including anti-p-IκBα, anti-IκBα, and anti-NFκB p65. GAPDH (Santa Cruz, #sc-32233) is chosen as housekeeping protein.\nRNA was extracted from THP1 cells using Trizol (Invitrogen). And total RNA was reversely transcribed using ProtoScript®II Reverse Transcriptase (NEB). Quantitative PCRs were performed triplicates for all samples using the Mx 3005P Real-Time PCR system (Stratagene) with SYBR Green I nucleic acid gel stain (Life Technologies). For normalization, ΔC t  values were calculated relative to the levels of Actin transcripts. Primers for qRT-PCR were listed in Table  S1 .\nImmunofluorescent was performed for TNF-α-stimulated nucleus translocation of NF-κB. The primary antibody used was anti-NFκB p65 (1:100 dilution; CST #8242). Secondary antibody is anti-rabbit IgG HRP-linked antibody (1:1000 dilution; CST #7074). Nuclei were revealed by DAPI staining. Fluorescence images were collected under confocal microscope system (Leica Microsystems, Wetzlar, Germany).\nFemale WT BalB/C mice (18–20 g) were obtained from Animal Facility at University of Macau. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Panel on Animal Research Ethics of University of Macau. Mice were acclimated for 1 week prior to use in experiments with free access to water and chow diet through the experiment.  In vivo  experiments were performed in the animal facility.\nBABL/c mice were randomly divided into three groups, sham, LPS and LPS plus compound  3b  treatment group. Mice in the treatment group were pre-treated with compound  3b  (45 mg/kg) orally for 9 days, once daily. Mice in sham and LPS group were given PBS administration as placebo. All the mice were anesthetized with isofluorane suspended from incisors. PBS or LPS (200 μg/ml in PBS, 100 μg/kg) were administered intragastrically 1 h after the oral administration from day 7 to day 9. The intragastrically administration with LPS or PBS were given three times per day for three days continuously. Mice were sacrificed 6 h after the last intragastrically administration to collect serum and lung tissue.\nMice were sacrificed after being anaesthetized with CO 2 . Serum was collected and stored at −70 °C until analysis. Lung was removed, fixed and saved for histologic analysis.\nAfter fixation, lung sections were stained with haematoxylin-eosin staining solution and examined under light microscopy. At least three different sections were examined per lung section.\nLung sections were fixed with paraffin, and washed by PBS before block with 3% H 2 O 2  solution. Paraffin-fixed lung sections were then washed by TBS-T and blocked in blocking buffer containing 10% goat serum, 0.1% BSA, 0.2% gelatin at room temperature for 1 hour. Lung sections were then incubated in primary antibody against MPO or phosphorylated p65 in blocking buffer at 4 °C overnight, washed by TBS-T and then incubated with HRP-conjugated secondary antibody. A set of slides were processed without incubation with primary antibody as negative control. The signal was detected by DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). The slides were counterstained with hematoxylin and mounted for immunohistochemistry analysis. The sections were incubated with FITC-conjugated secondary antibody and counterstained with DAPI for immunofluorescent analysis.\nAs a negative control, a set of slides was processed without primary antibody. To quantify stains of different proteins, pictures were taken of >30 fields of view at ×400 magnification. Adobe Photoshop CS2 software was used to pixel count the positive staining. The stains were scored by three researchers separately in blind.\nData are presented as mean ± S.D. or mean ± S.E.M. No animals were excluded for analysis. All experiments were repeated two or more times. Data were normally distributed, and the variance between groups was not significantly different. Differences in measured variables between groups were analyzed by one-way or two-way ANOVA, or the student’s  t  test by GraphPad Prism 5 software. Results were considered statistically significant when  p  < 0.05.\nExperimental details for the syntheses, NMR spectra and HPLC purity analysis of listed compounds, and general information for biological evaluation. This material is available free of charge via the Internet.\nAll data generated or analyzed during this study are included in this published article and its Supplementary Information files.\n\nSupplementary information\nSupplementary information","source_license":"CC-BY-4.0","license_restricted":false}