BuyangHuanwu Decoction Inhibits LPS-induced Inammation in BV2 Microglial Cells by NF-κB/NLRP3 Pathway BYHWD Inhibits LPS-induced Inammation in BV2 Cells

Background: Neuroinammation has been implicated in the pathogenesis of various neurological and cerebrovascular diseases. Recently, microglia has become a promising target to treat neuroinammation-related diseases. BuyangHuanwu Decoction (BYHWD), a traditional Chinese medicine recipe, is a representative prescription for the treatment of neuroinammation, showed great therapeutic potential in neuroinammation-related cerebrovascular disorders including cerebral ischemia. However, it’s a possible mechanism of action remains unclear. Methods: In the present research, we studied the therapeutic effects of BYHWD on LPS-induced inammatory response in BV2 microglia cells. The inammatory cytokines include IL-6 (cid:0) TNF-α (cid:0) NO were detected by the commercial kit. The inammatory-related protein was measured by western blot and immunouorescence. Results: Our ndings showed that BYHWD could signicantly reverse LPS-induced morphological changes in BV2 microglial and inhibits the local synthesis of proinammatory cytokines and free radical via directly regulating NF-κB activity and inammasome assembly. Our data showed that BYHWT inhibits LPS-induced TNF-α, IL-6, and NO production, Also can inhibit iNOS protein expression and the activation of NF-κB according to inhibit NLRP3 activation. Conclusions: In conclusion, our results demonstrated that BYHWD is a potential candidate for treating neuroinammation-related diseases. Certainly, more comprehensive researches on the precise mechanism of action of BYHWD in cerebrovascular disease is required in future vivo studies.


Introduction
Neuroin ammation has been implicated in the pathogenesis of various neurological and cerebrovascular diseases, such as Alzheimer's disease, depression, stroke and cerebral ischemia etc. 1,2 The immune response within the central nervous system (CNS) involves local immune cells and signaling pathways.
Microglia, which is the resident macrophage in the CNS, are versatile cells involving in triggering in ammatory pathways and oxidative stress 3 . In response to exogenous stimuli, activated microglia transmit in ammatory reactions by releasing pro-in ammatory cytokines and mediators. Thus, recently, microglia has become a promising target to treat neuroin ammation-related diseases.
BuyangHuanwu decoction (BYHWD) was originally recorded in the traditional herbal literature of Yi-Lin-Gai-Guo written by Qing-Ren Wang in 1830 during the Qing Dynasty. According to the Chinese Pharmacopoeia 4 , the decoction is comprised of seven commonly used Chinese herbal drugs: Radix Astragali (huangqi), the dried roots of Astragalus membranaceus (Fisch.) Bge. var., mongholicus (Bge.) Hsiao; . The carda part of Radix Angelicae Sinensis root (guiwei), the dried lateral roots of Angelica sinensis (Oliv.) Diels; . Radix Paeoniae Rubra (chishao), the dried roots of Paeonia lacti ora Pall.; d. Rhizoma Chuanxiong (chuanxiong), the dried rhizomes of Ligusticum chuanxiong Hort; .FlosCarthami (honghua), the dried owers of Carthamus tinctorius L.; .Semen Persicae (taoren), the dried seeds of Amygdalu spersica L.; and . Pheretima (dilong), the dried bodies of Pheretima aspergillum (E. Perrier), in the ratio of 120:6:4.5:3:3:3:3 on a dry weight basis, respectively. According to the traditional Chinese medical literature, this formula has been shown to provide neuroprotective effects for neuroin ammation related conditions such as brain ischemia 5,6 , stroke 7,8 . A previous study has found that BYHWT have the potential improvement for ischemic stroke and extended lifespan, primarily by regulating neuroin ammation, apoptosis, angiogenesis and blood coagulation, as well as by mediating neurogenesis and nervous system development 9 . However, the nothing is known about the effects of BYHWD in regulating neuroin ammation. Thus, in the present study, we focused on the effects of BYHWD on LPS-induced in ammatory response in BV2 microglia cell, and aimed to determine the possible mechanism underlying its anti-in ammatory action.

Drug Administration
Buyang Huanwu Decoction is a granule made up of Radix Astragali seu Hedysari, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Lumbricus, Semen Persicae, Flos Carthami, Rhizoma Ligustici Chuanxiong, according to the ratio of 120 : 6 : 4.5 : 3 :3 : 3 : 3.The decoction was made by boiling the mixture in distilled water at 100°C for 30 min three times, then the drug solution was cooled and dried to give the drug powder, weigh 400mg powder and dissolve in 40ml DMEM medium to make 10mg/ml mother liquor.

Drugs and Treatment
BYHWT was dissolved in DMEM medium to a concentration of 10mg/mL, LPS(Sigma) was dissolved in PBS(Procell) to a concentration of 1mg/mL, then pass 0.22um sterile lter. In all experiment, cells were pretreated with BYHWT(1-8mg/ml) for 2h before stimulated with LPS(400ng/mL) for 24h.

CCK-8 Assay
BV2 cells were seeded in 96-well plates at a density of 8*10 3 cells per well(100µL per well). The cells were processed as described above, and 6 parallel holes were set for each group. After 24 hours of stimulation, remove the cell supernatant and add medium containing 10% CCK-8(Biosharpe) and the culture was continued for 3h, The absorbance was measured with a microplate reader (5LX800,Biotek)at 450 nm.

NO Assay
BV2 cells were seeded in 96-well plates at a density of 8*10 3 cells per well(100µL per well). The cells were processed as described above, and 6 parallel holes were set for each group. After incubation for 24h, the supernatant of culture was collected and the concentration of NO was measured by commercial kit (Beyotime )according to the manufacturer's instruction.

ELISA Assay
BV2 cells were seeded in 6-well plates at a density of 1*10 5 cells per well(2mL per well). The cells were processed as described above, and 3 parallel holes were set for each group. After incubation for 24h, the supernatant of culture was collected, and analyzed by ELISA according to the manufacturer's protocol(MULTI SCIENCES) 2.7 Western Blotting BV2 cells were seeded in 6-well plates at a density of 1*10 5 cells per well (2mL per well), incubated and treated as mentioned above. Cultured cells were lysed with RIPA buff(add PMSF at 100:1),After lysing the cells on ice for 30 minutes, The supernatants were collected and then quantitated for protein determination using a BCA Protein Assay kit and the cells protein(30µg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2h at 80V,and transferred to polyvinylidene uoride (PVDF) membranes(Immobilon, USA) for 120 min at 200 mA. After blocking with 5% non-fat milk or BSA for 1h at 37℃, the membrane was incubated overnight with primary antibodies.
2.8 Immuno uorescence staining BV2 cells were seeded in 24-well plates at a density of 3*10 4 cells per well(500µL per well), incubated and treated as mentioned above. After xed with 4% paraformaldehyde for 20 min at room temperature, cell were permeabilized with 0.1% Triton X-100 for 20 min. Then blocking with 3% BSA for 1h at 37℃, and were incubated overnight at 4℃ with primary antibodies.

Statistical analysis
All analyses were performed using SPSS version 23.0 (IBM, USA). Data were represented as the means ± SEM. The data were analyzed with a one-way analysis of variance (one-way ANOVA) or repeatedmeasures analysis of variance (RM ANOVA) where statistically appropriate. When signi cant effects were detected, post hoc multiple pairwise comparisons were made with the LSD comparisons test after ANOVA. The analysis results were only reported when a signi cant difference was found. Statistical signi cance was de ned as P < 0.05.

Screening of the best dose for BWT and LPS
To determine the effect of BYHWT on the toxic effect of BV2 microglia, the CCK-8 assay was performed 24 h after treatment with various concentrations of BYHWT ranging from 1 mg/mL to 9mg/mL. Results (Fig1A) show that the dose of the drug shows obvious cytotoxicity at 9mg/ml. Thus, the concentration of BYHWT range of 1-8mg /mL for the subsequent experiments. According to Fig1B and Fig1C, 400ng/ml of LPS is not signi cantly toxic to cells, and has the highest level of NO production for cells. After the cells were pretreated with different concentrations of BYHWT for 2h, 400ng/mL LPS was added to stimulate the cells for 24h, the addition of LPS and BYWHT had no signi cant effect on cell viability(Fig1D), indicating that subsequent experiments can be performed at this concentration. As indicated in Fig1E, the morphological changes in BV2 cells were evaluated after treatment with BYHWT with or without LPS, microscopic showed that the resting microglia were spindle shaped with small cell bodies and long cellular pseudopods. After stimulated with LPS, the cells have reduced pseudopods, and cell bodies become larger and rounder, but BYHWT effectively reduced this change, And as the drug dose increases, the number of cells tending to a resting state increases. These results show that BYHWT can signi cantly decrease the change of cell morphology induced by LPS.

BYHWT inhibit LPS-induced TNF-α, IL-6,and NO production, And can inhibit iNOS protein expression
To investigate the anti-in ammatory effects of BYHWT, the expression of in ammatory mediators were detected in this study by ELISA.As shown in Fig2A and 2B,the IL-6,TNF-α levels increased in the cell culture media in the LPS treated alone group, and pre-treatment with different concentration of BYHWT dramatically decrease of cytokine production, same as NO production level (Fig2C).Similarly, iNOS protein from cells for Western Blot and Immuno uorescence detection also showed the same results (Fig2E).

BYHWT effectively inhibits LPS-induced activation of NF-κB
Page 6/10 NF-κB activation mediates many in ammatory responses in the central nervous system. To investigate the anti-in ammatory mechanism of BYHWT, Western blot analysis used to detect the protein expression of NF-κB and IκB-α expression level. As shown in Fig3A-B,the LPS stimulation resulted in phosphorylation levels of NF-κB p65 and IκB-α,and the effect were signi cantly inhibited by the treatment of BYHWT decrease of IκBα protein level and and IκBα increased signi cantly after compared with control group.
However, The molecular mechanisms of BYHWT on neuroin ammation involved signi cantly inhibited IκB phosphorylation and degradation following LPS activation 3.4 BYHWT decrease in ammatory response through inhibits NLRP3 activation To investigate whether BYHWT affects the NLRP3 in ammasome activation in LPS-activated BV2 cells, The expression protein levels of NLRP3 were examined by western blot analysis and Immuno uorescence detection. As shown in Fig4A-4B, the NLRP3 level were markedly elevated after LPS stimulation, and treatment with signi cantly decreased LPS-induced NLRP3 level in BV2 cells. It indicates that the anti-in ammatory effect of BYHWT may be achieved by regulating the activation of NLRP3.

Discussion
BuyangHuanwu decoction (BYHWD), a traditional Chinese medicine (TCM) prescription, has long been used clinically to aid neuroprotective effects after stroke. According to experimental reports, BHD improves blood circulation, controls pain, regenerates neuronal cells [10][11][12] .And protects against the neuronal damage caused by ischemic and oxidative stress 13,14 .BYHWT have been traditionally used to treat cognitive de cits and other brain disorders in TCM owing to the virtues of neuroprotection, antioxidative stress anti-apoptosis, anti-in ammation and neurotrophic actions 5,14-17 .Our research it is, or at least partially, helpful to better comprehend the effective underlying mechanisms of classical prescription.
In the present research, we found that BYHWD was effectively to inhibit LPS induced in ammatory responses in BV2 microglia cells. We noted that BYHWD under 9 mg/ml showed no toxicity to BV2 microglial cells. It could signi cantly reduce LPS-induced morphological changes and inhibit the production of pro-in ammatory cytokines and free radical. LPS is the most widely used in ammatory mediator, which can activate microglia and trigger the pro-in ammatory signaling cascade In the present research, we found that BYHWT was effectively to inhibit LPS induced in ammatory responses in BV2 microglia cells. We noted that BYHWT showed no toxicity to BV2 microlial cells. It could signi cantly reduce LPS-induced morphorlogical changes and inhibit the production of pro-in ammtory cytokines and free radical. LPS is the most widely used in ammatory mediator, which can activate microglia and trigger the pro-in ammatory signaling cascade 18 . In normal states, microglia surveils the microenvironment whilst maintains the homeostasis in the brain. While in disease states, in response to injury, harmful toxins, infection or in ammation, microglial cells secret pro-in ammatory mediators, which are regulated by the transcription factor nuclear factor kappa B (NF-κB), to amplify neuroin ammation and result in pathological changes. NF-κB is the central regulator of inlammation that controls the gene transcription of chemokines, cytokines, proin ammatory enzymes, adhesion molecules and proin ammatory transcription factors 19,20 . Consistently, we observed that NF-κB was activated in BV2 microglia cells and resulted in the generation of proin ammatory cytokines (IL-6 and IL-1β) and inducible enzymes (iNOS), as well as nitric oxide (NO). In resting cells, the inhibitors of κB (IκB) family sequesters NF-κB in the cytoplasm. LPS activates a complex of IκB kinases (IKK) and results in the phosphorylation of IκB protein, which is rapidly ubiquitinated and degraded 21 . This alteration leads to the phosphorylation of NF-κB p65 at Ser536 and the release of NF-κB from IκB. Liberated NF-κB then translocates to the nucleus and binds to speci c gene promoter elements to initiate transcription 22 .
Therefore, the regulation of NF-κB is crucial in neuroin ammation-relatted diseases control 23 . Our data displayed that SYG was effective in regulating the activity of NF-κB.
Moreover, activated NF-κB enhances the transcriptional level of cytosolic innate immune signaling receptor NOD-, LRR-and pyrin domain-containing 3 (NLRP3), a pivotal mediator of IL-6-associated neuroin ammation 24,25 .Targeting NLRP3 pathway is also a promising strategy to develop pharmacotherapy for in ammation-associated diseases. Our nding indicated that BYHWT could inhibit local synthesis of IL-6 by suppressing the formation of NLRP3 in ammasome and overexpression.
In conclusion, our results demonstrated that BYHWD is a potential candidate for treating neuroin ammation-related diseases via directly regulating NF-κB activity and in ammasome assembly. Certainly, more comprehensive researches on the precise mechanism of action of BYHWD in cerebrovascular disease is required in future vivo studies.

Conclusions
BYHWT inhibits LPS-induced TNF-α, IL-6, and NO production, Also can inhibit iNOS protein expression and the activation of NF-κB according to inhibit NLRP3 activation. Our ndings provided a new strategy that BYHWD is a potential candidate for treating neuroin ammation-related diseases via directly regulating NF-κB activity and in ammasome assembly.