Isoforskolin and forskolin attenuate lipopolysaccharide‐induced inflammation through TLR4/MyD88/NF‐κB cascades in human mononuclear leukocytes

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll‐like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF‐κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF‐κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL‐2, IL‐6, IL‐21, IL‐23, tumor necrosis factor (TNF) α, and TNF‐β were tested by enzyme‐linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF‐κB in MLs and the production of IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF‐κB, IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down‐regulating protein levels of IL‐1β and TNF‐α, in which TLR4/MyD88/NF‐κB signal pathway could be involved.


| INTRODUCTION
The plant Coleus forskohlii is distributed primarily in India, Thailand, China, Egypt, and Brazil and has a history of use in the treatment of multiple diseases. Isoforskolin (ISOF), also named 6-acetyl-7-deacetyl forskolin, was isolated from C. forskohlii, a tropical perennial plant native to Yunnan in China (Yang et al., 2011). It is reported that the Yunnan native plant does not mainly contain forskolin (FSK) but ISOF, which has attracted attention for its biological functions. Our previous study demonstrated that ISOF activated adenylyl cyclases (ACs) and then increased cyclic adenosine monophosphate (cAMP) levels in rat liver homogenate, and it also relaxed the contraction of isolated guinea pig lung and trachea smooth muscle induced by histamine (Wang & Cao, 2013). Moreover, pretreatment with ISOF attenuated acute lung injury (ALI) in animal models and decreased the production of inflammatory cytokines tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL-6, and IL-8 (Yang et al., 2011). One of our members' research indicated that ISOF down-regulated the transcription and expression of TNF-α and IL-6 in murine macrophages, human macrophages, and dendritic cells induced by recombinant Borrelia burgdorferi basic membrane protein A and may have a potential clinical application as an anti-inflammatory agent for the treatment of Lyme arthritis (Zhao et al., 2017).
ISOF is an analog of diterpene FSK, ISOF is extracted from C. forskohlii distributed in Yunnan, China, and FSK is extracted from C. forskohlii distributed in India. It is well known that FSK is an effective AC activator and causes an increase in intracellular cAMP. Many researches showed that cAMP-induced modulation of the receptor for advanced glycosylation end products isoform in macrophages can control the inflammatory state in both in vitro and in vivo experimental conditions (Motoyoshi et al., 2014). FSK has effects on antiinflammation through cAMP signaling (Sousa et al., 2010;You, Xiong, Zhang, Shi, & Shi, 2017). Our previous study also showed ISOF activated ACs, increased cAMP, and then attenuated inflammation in ALI induced by lipopolysaccharide (LPS; Yang et al., 2011). Furthermore, another study showed AC activation attenuated transmembrane toll-like receptor 4 (TLR4) signaling in a murine macrophage cell line (RAW264.7) and bone marrow-derived macrophages when stimulated with LPS (Cai, Du, Feng, et al., 2013). Therefore, we hypothesized that ISOF and FSK activated ACs and then regulated TLR4 signaling.
TLR4 mediates the inflammatory process evoked by LPS (Hu, Lou, Mao, et al., 2016). Myeloid differentiation factor 88 (MyD88) is a tolllike receptor (TLR) adaptor. After recognition by TLR4, a series of cascades including MyD88 are initiated, and then MyD88 primarily activates nuclear factor kappa B (NF-κB) family members and mitogen-activated protein kinase (Liu & Ding, 2016). Activation of NF-κB has a variety of biological functions such as control DNA transcription, cytokine production and cell survival time, and extensive involvement in the body's immune system, inflammation, and stress physiological pathology (Baker, Hayden, & Ghosh, 2011). Therefore, inhibition of NF-κB activity is an effective way to block inflammation.
On the basis of the above factors and to better understand biological effects of ISOF, we hypothesized that ISOF and FSK pretreatment attenuated inflammatory reaction through down-regulation inflammatory factors such as the protein levels of TNF-α, IL-1β, and other inflammatory factors in supernatant of mononuclear leukocytes (MLs) induced by LPS, and the mechanism of anti-inflammation of ISOF partly related to TLR4/MyD88/NF-κB signal pathway.

| Protocols in human peripheral blood mononuclear cells
Human peripheral blood mononuclear cell samples from healthy volunteers were kindly supplied by the First Affiliated Hospital of Kunming Medical University. The hospital ethics committee approved the study and all the volunteers provided informed consent. Human MLs were isolated by using density gradient centrifuge (Fuss, Kanof, Smith, & Zola, 2009;Kanof, Smith, & Zola, 2001). The procedures yield a 95-98% viable MLs (by trypan dye exclusion) and 95% pure MLs population (by morphology in Giemsa stains). Cells were washed by phosphate-buffered saline buffer and resuspended in Roswell Park Memorial Institute 1640 medium containing 1% bovine serum albumin, and cell count was adjusted to 5 × 10 6 /ml for use. MLs were suspended in 6-well plates (2 ml/well); preincubated with at 37°C (5% CO 2 ) for 2 hr; given pretreatment with ISOF (1.0 μM), FSK

| Measurement of TNF-α and IL-1β by ELISA
Protein levels of TNF-α and IL-1β in ML supernatants were assayed using a commercially available ELISA kit (human TNF-α and IL-1β ELISA, R&D, USA). A polyclonal antibody specific for TNF-a and IL-1β was used for coating the 96-well microtiter plates. Briefly, 100 ml of the different standards was added into the appropriate wells in duplicate. One hundred milliliter of supernatants was added in duplicate into additional wells. The plate was covered and incubated on the constant temperature culture shaker for 2 hr at 25°C. After extensive washing to remove unbound compounds, TNF-α and IL-1β was recognized by the addition of 100 ml of polyclonal antibody specific for TNF-α and IL-1β (detection antibody), and then the plate was covered and incubated on the constant temperature culture shaker for 1.5 hr at 25°C. After removal of excess polyclonal antibody and repeated washing to remove unbound compounds, 100 ml of horseradish peroxidase-conjugated antirabbit IgG (secondary antibody conjugate) was added, and the plate was covered and incubated on the constant temperature culture shaker for 0.5 hr at 25°C. Subsequently, the bound peroxidase activity was quantified using 50 ml of the substrate 3,39,5,59-tetramethyl benzidine. The color reaction was developed at ambient temperature in the dark for 10 min. The intensity of the color reaction was measured at 450 nm after acidification and was directly proportional to the concentration of TNF-α and IL-1β in the standards. According to a standard curve established using recombinant TNF-α and IL-1β, the concentration of TNF-α and IL-1β in individual samples were calculated. The minimum detectable dose of IL-1β is typically less than 1.0 pg/ml. The minimum detectable dose of TNF-α is typically less than 7.8 pg/ml.

| Western blot analysis
The TLR4, MyD88, and NF-κB protein levels in lysed cell were examined by western blot analysis. Protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, Jiangsu, China). Total protein (20 μg) was fractionated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 10% defatted milk powder solution at room temperature for 2 hr and incubated overnight at 4°C with the rabbit antibodies against TLR4 (concentrate on 1:500), MyD88 (concentration 1:700), NF-κB (concentration 1:700), GAPDH (concentration 1:10,000), and beta-actin (concentration 1:2,000). After three washes, membranes were incubated with horseradish peroxidase-conjugated goat antirabbit IgG antibody for 1.5 hr at room temperature. Then, washing was repeated four times, and the protein was visualized with an enhanced chemiluminescence kit (Sigma, USA).
The density values of bands were quantified by densitometric analysis of scanned images (Scion Image 4.03). The relative protein ratio was calculated by determining the integrated intensity of the bands of each treated group as a ratio of the control condition.

| Statistical analysis and calculations
Data was expressed as means ± SEM. Statistical analysis was performed using statistical software Sigma Stat 3.5. Comparisons were made using one-way analysis of variance, followed by Student-Newman-Keuls (SNK) test analysis if there are normal distribution and variance homogeneity of data; one-way analysis of variance on rank was used if there is no homogeneity of data variance. p < 0.05 was considered statistically significant. The graph was performed using software Sigma plot10.

Cytokines of ML supernatants detected by Quantibody Human
TH17 Array 1 were scanned by using InnoScan 300 Microarray Scanner and were analyzed by using Quantibody Human TH17 Array Data analysis software.

| Effects of ISOF/FSK on cytokine expression in ML supernatants by cytokine array analysis
All 20 cytokines were detected in control, LPS, ISOF, FSK, RF, and DM groups. As seen from Table 1,  3.2 | Effects of ISOF/FSK on TNF-α and IL-1β in human ML supernatants by ELISA assay In order to verify the results of quantification of cytokine analysis, TNF-α and IL-1β in ML supernatants were tested by the ELISA method. As seen from Figure 2a

| Effects of ISOF/FSK on TLR4, MyD88, and NF-κB in human MLs
TLR4 is a transmembrane protein, member of the TLR family, which belongs to the pattern recognition receptor family. Its activation can activate MyD88 and can then lead to an intracellular signaling pathway NF-κB and inflammatory cytokine production.
The content of key molecules of TLR4 pathway was quantified by western blot. As seen in Figure 3a Our result shows that ISOF attenuates inflammation in MLs induced by LPS through down-regulating protein expression of inflammatory factors. Furthermore, ISOF has the following effects: suppressing ocular hypertension in rabbits (Li et al., 2000); increasing cAMP levels in rat liver homogenate; relaxing the contraction of isolated guinea pig lung and trachea smooth muscle induced by histamine (Wang & Cao, 2013); attenuating ALI in animal models induced by    respectively for half an hour prior to LPS (1 μg/ml) treatment for 6 hr. MLs supernatants were collected for detection of cytokines. Log C is derived from raw data (pg/L) through logarithmic transformation. interleukin (IL)-1β, IL-2, IL-6, IL-21, IL-23, and tumor necrosis factor (TNF)-β were analyzed by one-way analysis of variance (ANOVA), whereas TNF-α and transforming growth factor-β1 were analyze by one-way ANOVA on rank. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS group. Data are means ± SEM; n = 4 repeat experiments with the same volunteer that both ISOF and FSK inhibit inflammatory response in MLs induced by LPS, but the relationship between them is not known and need to be further studied.
Numerous researches show that FSK is an effective AC activator and causes an increase in intracellular cAMP and then plays the effects of anti-inflammation (Sousa et al., 2010;You et al., 2017). This study is similar with our results. Moreover, FSK has effects of weight loss ( Other than NF-κB p65, other pro-inflammatory cytokines such as IL-1β, IL-2, IL-6, IL-8, and TNF-a were important mediators of the inflammatory response in ALI/ARDS (Mokra & Kosutova, 2015), In ALI/ARDS, IL-1β, TNF-α, IL-8, and IL-6, which are usually increased, in the bronchoalveolar lavage fluid of patients at risk of ARDS or with established ARDS, IL-1β, TNF-a (Park, Goodman, Steinberg, et al., 2001), and IL-8 (Gonzalez-Lopez, Garcia-Prieto, Batalla-Solis, et al., 2012) were elevated. Additionally, high concentrations of IL-6 may serve as a predictive marker of poor outcome (Bouros, Alexandrakis, Antoniou, et al., 2004). Our detecting results of quantification of cytokine analysis chip showed that the protein levels of IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-α, and TNF-β in the LPS group was higher than the control group in supernatants of MLs, but pretreatment with ISOF, FSK, and DM, respectively, showed that the effects of LPS on MLs were inhibited. This indicated that the ISOF, FSK, and DM have antiinflammation effect on MLs induced by LPS through adjusting the expression of IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-a, and TNF-β, but that some cytokines needed to be further identified by experiments.
In the present study, except for the RF group, the change trend of protein levels of TNF-α and IL-1β detected by ELISA and quantification of cytokine analysis were similar in other experimental groups. The value of cytokines detected by quantification of cytokine analysis chip in the RF group were higher than that in the LPS group, the reason might be that the dose of RF may have been too high for that person. , respectively for 0.5 h, and then each group was given treatment with LPS (1 μg/ml) for 6 hr. One-way analysis of variance followed by SNK was used to process the data of TNF-α and IL-1β. * p < 0.05, **p < 0.01, and *** p < 0.001, compared with LPS group. Data are means ± SEM; n = 5 independent experiments with independent volunteer In addition, we also found that DM was able to attenuate expression of inflammation cytokines NF-κB p65, TNF-α, and IL-1β in MLs induced by LPS, but it could not effectively reduce the protein level of TLR4 and MyD88. The reasons may be that DM had antiinflammation effect on MLs induced by LPS related to another pathway, but TLR4/MyD88 pathway.
In conclusion, the results of this study revealed that ISOF and FSK attenuate inflammation in MLs induced by LPS through downregulating protein levels of pro-inflammatory cytokines IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-α, and TNF-β, in which TLR4/MyD88/NF-κB signal pathway could be involved. These findings suggest that ISOF and FSK could be a potential candidate for the treatment of inflammation induced by LPS.

FUNDING INFORMATION
This work was partially supported by the National Natural Science  , DM (25 μM) respectively for 0.5 h, and then each group was given treatment with LPS (1 μg/ml) for 6 hr. One-way analysis of variance (ANOVA) followed by SNK was used to process the data of TLR4 and NF-κB. One-way ANOVA on rank followed by SNK was used to process the data of MyD88. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS group. Data are means ± SEM; n = 5 independent experiments with independent volunteer

CONFLICTS OF INTEREST
The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors state no conflict of interest.