| Nerve, endocrine and immune systems are interrelated with each other to constitute a complex neuroendocrine immune network (NEI) and keep body health. a-Melanocyte stimulating hormone (?-MSH) as an endogenous neuroimmunomodulation peptide, can affect different cells of immune system and neuroendocrine system through melanocortin receptor (MCRs) to reduce the production of inflammatory cytokines and activate the anti-inflammatory response. The significant anti-inflammatory effect of?-MSH has been proved through a number of animal experiments, but the effect mechanism has not been fully elucidated.In the process of inflammatory response induced by LPS, the production of TNF-?is mainly through IKK/IKB/NF-?B (NF-?B) pathway and Raf-1/MEK1-MEK2/ERK1-ERK2 (ERK) pathway. In the NF-?B signaling pathway, IKK plays a very important role, although the upstream signal path is different. IKK?, a subunit of IKK, plays an important role in phosphorylating I?Ba and the regulation of classic, non-classical NF-?B signaling pathway. Therefore, IKK?is considered to be an important subunit of IKK. In the ERK pathway, activation of Raf-1 is able to activate its downstream mitogen-activated protein kinase kinase (MEK) and mitogen-activated protein kinase (MAPK), and further activate the downstream transcription factors, which promote the production of TNF-a. ERK pathway is also coupled NF-?B signaling pathway. When S6 kinase (p90), the ERK pathway downstream component, is activated, it enables the phosphorylation and degradation of I?B?and leads to NF-?B release into the nucleus, then promotes TNF-a production.MicroRNAs are short RNA molecules with 18-23 nucleotides that regulate the post-transcriptional expression of their target genes, and play a critical role in health and many diseases. Although studies have shown that a-MSH inhibits these two signaling pathways, as mentioned above, directly or indirectly by regulating PKA, P38 kinase, I?B?and so on, the changes of IKK?and Raf-1 as well as related microRNA regulatory mechanisms under the stimulation of a-MSH and LPS have not been elucidated. Therefore, the aim in this study was to investigate the mechanisms of the changes of IKK?and Raf-1 as well as the actions of related microRNAs under the stimulation of?-MSH and LPS.Section One:?-MSH inhibits TNF-a production induced by LPS and upregulates the expression of hsa-miR-15b and hsa-miR-199a in THP-1 cell lineFirstly, to evaluate the inhibition of?-MSH on TNF-?production induced by LPS, we stimulated THP-1 cells with 5×10-8mol/L?-MSH,50 ng/ml LPS,5×10-8 mol/L?-MSH+50 ng/ml LPS, respectively. The experimental groups are as follows: control group,?-MSH group, LPS group,?-MSH+LPS group. Enzyme-linked immunosorbent assay (ELISA) was used to test TNF-?in the supernatants from the four groups. The relative TNF-?mRNA expression was tested by Real-Time qPCR. The results of ELISA showed that TNF-?level in?-MSH+LPS group was significantly lower than that of LPS group (P<0.01), indicating that?-MSH could inhibit LPS-induced TNF-?production. Real-Time qPCR results also showed that TNF-?mRNA expression of?-MSH+LPS group was significantly lower than that of LPS group (P<0.01), suggesting that?-MSH could inhibit TNF-?mRNA expression induced by LPS.Secondly, we selected IKK?and Raf-1 as the respective target genes of hsa-miR-199a and hsa-miR-15b by targetscans and MiRBase prediction methods. To investigate whether hsa-miR-15b and hsa-miR-199a exist in THP-1 cells, and whether?-MSH and LPS affect the expression of these microRNAs, we set up the control group,?-MSH group, LPS group and the?-MSH + LPS group, and Real-Time qPCR was used to detect hsa-miR-15b and hsa-miR-199a relative expression in the four groups. The results showed that hsa-miR-15b and hsa-miR-199a existed in THP-1 cells. a-MSH increased their expression (compared with the control group, P<0.01), while LPS had an inhibitory action (compared with the control group, P<0.01); hsa-miR-15b and hsa-miR-199a expression in?-MSH + LPS group was significantly higher than that of LPS group (P<0.01), suggesting that?-MSH could promote the expression of hsa-miR-15b and hsa-miR-199a and antagonize the inhibitory effect of LPS.The above results indicate that hsa-miR-15b and hsa-miR-199a exist in THP-1 cells. Both?-MSH and LPS affect their expression. Then, we further investigated whether they also exist in normal human peripheral blood mononuclear macrophages (PBMCs), and whether their expression is regulated by a-MSH and LPS.Section Two:a-MSH inhibits TNF-a production induced by LPS and upregulates the expression of hsa-miR-15b and hsa-miR-199a in human peripheral blood mononuclear macrophagesFirstly, to study the dose-response relationship between LPS and TNF-a production in PBMCs, PBMCs were stimulated by different concentrations of LPS (0, 1,10,100,1000 ng/ml) for 6 h. To test the time-response relationship of TNF-a production induced by LPS, TNF-a was detected at the different time points (0 min,30 min,45 min,1 h,2 h,4 h,6 h and 8 h) after 20 ng/ml LPS stimulation. To investigate the effects a-MSH on LPS (20 ng/ml) induced TNF-a produaction, PBMCs were treated by a-MSH with different concentrations (10-13,10-12,10-11,10-10,10-9,10-8 mol/L). The content of TNF-a in the supernatants was examined by ELISA. The results showed that the production of TNF-a was increased with the increase of LPS concentration, and the strongest effect of LPS stimulation was at 1000 ng/ml, indicating a dose-dependent manner in TNF-?production of PBMCs induced by LPS. TNF-a production began at 45 min after the stimulation of LPS (20 ng/ml), increased sharply between 2-6 h, then decreased gently after 8 h, but also kept a higher level. a-MSH (10-12?10-8 mol/L) could inhibit TNF-?production in PBMCs induced by 20 ng/ml LPS for 2 h, and 10-8mol/L?-MSH could completely inhibit TNF-?production stimulated by 20 ng/ml LPS.Secondly, the expression of hsa-miR-15b and hsa-miR-199a in PBMCs was tested using Real-Time qPCR, and the variation of hsa-miR-15b and hsa-miR-199a was examined at the different time points (15min 30min?1h?2h?4h?8h) of?-MSH or LPS stimulation. The experimental groups were same as that mentioned above: control group,?-MSH group, LPS group and?-MSH + LPS group. The results from Real-Time qPCR showed that hsa-miR-15b and hsa-miR-199a existed in PBMCs. In?-MSH group, hsa-miR-15b increased from 15 min to 8 h, and hsa-miR-199a peaked at 4 h and gradually decreased. In LPS group, hsa-miR-15b and hsa-miR-199a declined all the time. An upward trend of hsa-miR-199a expression in?-MSH + LPS group was observed 4h after LPS stimulation and consistent with that in a-MSH group, but the relative expression level was lower than?-MSH group.At the same time, we also used Real-Time qPCR and Western blotting to test the mRNA and protein expression of Raf-1 and IKK?in PBMCs treated by?-MSH, LPS and?-MSH+LPS, respectively. Real-Time qPCR results showed that?-MSH could inhibit the expression of Raf-1 mRNA (vs control, P<0.01), but had no effect on IKK?mRNA, while LPS could upregulate the mRNA expression of Raf-1 and IKK?(vs control, P<0.01). Raf-1 mRNA expression in?-MSH + LPS group was significantly lower than that in LPS group (P<0.05), indicating that?-MSH can antagonize the upregulatory effect of LPS on Raf-1 mRNA. The results of Western blotting showed that?-MSH could inhibit the protein expression of Raf-1, but had no effect on IKK?, while LPS could upregulate both Raf-1 and IKK?protein expression.The above results indicate that hsa-miR-199a and hsa-miR-15b exist in PBMCs, and both?-MSH and LPS can affect their expression.?-MSH only inhibits the mRNA and protein expression of Raf-1, but has no effect on IKK?. LPS promotes the mRNA and protein expression of Raf-1 and IKK?. The LPS-stimulated upregulation of Raf-1 mRNA can be antagonized partially by?-MSH.Section Three:Target identification of hsa-miR-15b and hsa-miR-199a and their effects on TNF-?production induced by LPSFirstly, to further study whether Raf-1 and IKK?are the targets of hsa-miR-15b and hsa-miR-199a, respectively, we transfected the mimics, mimics control, inhibitor, inhibitor control of hsa-miR-15b and hsa-miR-199a into PBMCs, respectively. Western blotting and Real-Time qPCR were used to test the protein and mRNA expression of Raf-1 and IKK?.The experimental groups were as follows:mimics group, mimics control group, inhibitor group, inhibitor control group. Western blotting results showed that hsa-miR-15b or hsa-miR-199a mimics could inhibit the expression of Raf-1 or IKK?proteins, compared with mimics control group. hsa-miR-15b or hsa-miR-199a inhibitor could promote the expression of Raf-1 or IKK?protein, compared with inhibitor control group. Real-Time qPCR results showed that hsa-miR-15b mimics could inhibit the Raf-1 mRNA expression, compared with mimics control group (P<0.01), and hsa-miR-15b inhibitor could promote the Raf-1 mRNA expression, compared with the inhibitor control group (P<0.01), but the mimics and inhibitor of hsa-miR-199a had no effects on IKK?mRNA expression, suggesting that hsa-miR-15b inhibits Raf-1 by reducing its mRNA expression, while hsa-miR-199a inhibits IKK?in translation.Secondly, we constructed the 3'UTR of Raf-1 or IKK?into the fluorescent reporter plasmid vector. Hsa-miR-15b and Raf-1 3'UTR luciferase reporter gene vector were co-transfected into 293T cells. hsa-miR-199a and IKK?3'UTR luciferase reporter gene vector were also co-transfected into 29.3T cells. Using dual fluorescence report detection system, we determined whether hsa-miR-15b or hsa-miR-199a interacts with the Raf-1 3'UTR region or IKK?3'UTR region. The results of dual-luciferase reporter assay showed that hsa-miR-15b and hsa-miR-199a could interact with Raf-1 and IKK?3'UTR region, respectively. These results indicate that Raf-1 and IKK?are the targets of hsa-miR-15b and hsa-miR-199a, respectively.To further study the effects of hsa-miR-15b and hsa-miR-199a on LPS-induced TNF-a production, the mimics and inhibitor of hsa-miR-15b and hsa-miR-199a were transfected into PBMCs, respectively. Then, the cells were stimulated by LPS for 4 h. ELISA was used to test TNF-a in supernatants, and Real-Time qPCR was used to test TNF-a mRNA. The experimental groups were as follows:mimics control group, hsa-miR-15b mimics group, hsa-miR-199a mimics, hsa-miR-15b mimics+ hsa-miR-199a mimics group, and the inhibitor control group, hsa-miR-15b inhibitor group, hsa-miR-199a inhibitor group, hsa-miR-15b+hsa-miR-199a inhibitor group. ELISA results showed that the secretion of TNF-a in hsa-miR-15b mimics group, hsa-miR-199a mimics group, hsa-miR-15b+hsa-miR-199a mimics group were all significantly lower than that in mimics control group (compared with mimics control, hsa-miR-15b mimics group, P<0.05; hsa-miR-199amimicsgroup, P<0.05; hsa-miR-15b+hsa-miR-199a mimics group, P<0.01). The secretion of TNF-a in hsa-miR-15b inhibitor group, hsa-miR-199a inhibitor group, hsa-miR-15b+ hsa-miR-199a inhibitor group were all significantly higher than that in inhibitor control group (compared with inhibitor control, each of the P values were <0.05). Real-Time qPCR results also showed that TNF-a mRNA expression in hsa-miR-15b mimics group, hsa-miR-199a mimics and hsa-miR-15b+hsa-miR-199a mimics group were all significantly lower than that in mimics control group (compared with mimics group, each of the P values were <0.01). TNF-a mRNA expression in hsa-miR-15b inhibitor group, hsa-miR-199a inhibitor group and hsa-miR-15b+hsa-miR-199a inhibitor group were all significantly higher than that in inhibitor control group (compared with inhibitor group, each of the P values were <0.01). The results indicate that hsa-miR-15b and hsa-miR-199a can inhibit TNF-?production induced by LPS.In conclusion, a-MSH promotes the expression of hsa-miR-15b and hsa-miR-199a, while LPS has an inhibitory effect. a-MSH inhibits Raf-1 expression by upregulating hsa-miR-15b to block ERK pathway, while LPS downregulates hsa-miR-15b and hsa-miR-199a to enhance the expression of Raf-1 and IKK?, and then promote the production of TNF-a. This study reveals a microRNA modulatory mechanism related with anti-inflammatory effect of a-MSH, which provides a new insight to elucidate the roles of NEI regulation network in the pathogenesis, prevention and treatment of the diseases, and suggests a new target for the research on anti-inflammatory drugs. |
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