Dahuang Zhechong Pills Suppress Silicosis Fibrosis Progression via p38 MAPK/TGF-β1/Smad Pathway In Vitro

Background Dahuang Zhechong pills (DHZCP) is a classic Chinese medicinal prescription in “Treatise on Cold Pathogenic and Miscellaneous Diseases (Shanghan Zabing Lun),” and it has the function of tonifying blood, nourishing Yin, and removing blood stasis. Previous studies have shown that DHZCP could alleviate SiO2 induced pulmonary fibrosis in mice. This study aims to further explore the preventive and therapeutic effects of DHZCP against silicosis fibrosis and the underlying mechanisms in vitro. Methods We used the experimental model of SiO2-induced MH-S cells to evaluate the therapeutic potential of DHZCP. MH-S cells induced by SiO2 were intervened with the drug-containing serum of DHZCP, and the effects of drug-containing serum of DHZCP on the MH-S cells were detected by CCK8, ELISA, flow cytometry, western blot, and immunofluorescence. Results DHZCP improved cell viability by reducing apoptosis. It also decreased the levels of TNF-α, IL-1β, IL-6 in the supernatant of MH-S cells induced by SiO2, inhibited the expression of p38 MAPK, blocked the activation of NF-κB, and controlled the upstream inflammatory response by multiple targeting. Concomitantly, we observed upregulation of Smad7 and a marked decline in TGF-β1, α-SMA, Smad2, Smad3 expression in MH-S cells treated with DHZCP. Conclusion To sum up, we conclude that DHZCP protects against SiO2-induced silicosis by reducing the persistent irritation of inflammation, regulating the p38 MAPK/TGF-β1/Smad pathway.


Introduction
Silicosis, one of the most common occupational diseases in the world is an irreversible and progressive interstitial pulmonary disease caused by long-term inhalation and retention of free silica (SiO 2 ) [1][2][3]. Despite the continuous exploration and progress in the prevention of silicosis, there were still many new cases every year [4]. is disease has brought a huge social and economic burden to the world, especially to developing countries [5]. e pathological processes of silicosis start from the phagocytosis of SiO 2 by macrophages, which leads to an inflammatory cascade, excessive proliferation and migration of fibroblasts, and gradual development of fibrosis [6]. Its pathological features include early infiltration of inflammatory cells, persistent pulmonary inflammation, excessive deposition of extracellular matrix (ECM), interstitial fibrosis, and formation of silicon nodules [7].
ough the pathogenesis and prevention of silicosis have been explored for many years, the exact and crucial pathogenesis has not been fully elucidated [8,9]. In terms of treatment, there are no specific targets for screening and diagnosis of silicosis in the early stage. Once silicosis is diagnosed, it is in the late stage of irreversible fibrosis, and effective drugs for its treatment are still lacking [6]. erefore, it is necessary to find the potential prevention and therapeutic methods and drugs of silicosis intervention [9].

Preparation of Drugs and SiO
Pyrrolidine dithiocarbamate ammonium (PDTC, CAS: 5108-96-3) is an NF-κB inhibitor. It was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Its purity is 99.86% and the specification is 10 mM * 1 mL in DMSO. 75 μL DMSO was added into 50 μL PDTC (10 mM), and after diluting and mixing, a stock solution with a concentration of 4 mM was obtained and stored at −20°C for standby.
SiO 2 was purchased from RHAWN (CAS: 60676-86-0, Shanghai, China), and the purity of the product was above 99%. e SiO 2 dust was dried at 180°C for 2 h, and a suspension of SiO 2 with a concentration of 20 mg/mL was prepared by mixing SiO 2 dust with sterile phosphate-buffered saline, which was then sterilized at 121°C for 20 min and then stored at −20°C. e suspension was then diluted to 40 μg/mL before use.

Preparation of DHZCP-Medicated Serum (DMS).
A total of 24 Sprague-Dawley male rats (SPF class; aged 5 weeks, 145-160 g) were purchased from the Chengdu Dashuo Laboratory Animal Co., Ltd. (Sichuan, China). All rats were raised in an air-conditioned room under stable temperature (23 ± 2°C) and humidity (50%-70%) with a regular 12 h light/dark cycle. e animals had unlimited access to tap water and standard food. e animal experiment in this study has been approved by the experimental animal ethics committee of Chengdu University of traditional Chinese medicine (Sichuan, China), and is in line with the guidelines for experimental animal care and use issued by the National Institute of Health. e rats were divided into 2 groups randomly: DHZCP and control group, with 12 rats in each group. e dose for the DHZCP group was 1.35 g/kg, and the control group was given an equal volume of distilled water. Rats were fed DHZCP by gavage twice a day for 7 days. After the last gastric perfusion for 1 h, all rats were anesthetized by sodium pentobarbital. Blood was collected from the abdominal aorta of rats in a sterile environment, stood 10 min (room temperature), and then centrifuged for 15 min at 2000 r/min. e supernatant was separated and inactivated at 56°C for 30 min, filtered with 0.22 μm microporous membrane, the DHZCP-medicated serum (DMS) was obtained. No adverse events occurred during the experiment.

Preparation of Reference Standard Solution.
Accurately weigh amygdalin, baicalin, catalpol, emodin, and paeoniflorin standard products, add appropriate amounts of pure methanol to them, and prepare a stock solution with a final concentration of 2 mg/mL. en use pure methanol stock solution to prepare these 5 analytes into a 500 ng/mL mixed standard, and start the sample injection analysis.  Table 2. e test was repeated 3 times. Data chromatogram acquisition and integration of high-resolution liquid/Mass Combination were processed by Xcalibur 4.1 ( ermo), and linear regression was performed with an equal weighting coefficient.

Cell Grouping, Poisoning, and Treatment.
e cells were divided into 5 groups: model control group [13] (MC, SiO 2 suspension, 40 μg/mL), DMS + PDTC group (40 μg/mL SiO 2 suspension +15% DMS + 4 mM PDTC), DMS group (40 μg/ mL SiO 2 suspension +15% DMS), PDTC group (40 μg/mL SiO 2 suspension +4 mM PDTC), and normal control group (NC, 15% drug-free serum). e cells were inoculated into 6-well plates with 5 × 10 5 cells per well, and then the culture medium containing FBS was replaced by the medium without serum. 4 mM PDTC was added into DMS + PDTC group and PDTC group, and the mixture was rested for 12 h. And then, SiO 2 suspension was added to the cells in the MC, DMS + PDTC, DMS, and PDTC with a final concentration of 40 μg/mL. Each plate was shaken gently and evenly and incubated for 4 h, 15% DMS was added to DMS + PDTC group and DMS group, respectively, after continuous cultivation for 12 h, the cells were taken out for subsequent experiments.

Cell Viability Was Measured. 4 mM PDTC was added
into DMS + PDTC group and PDTC group and the mixture was rested for 12 h. In addition to the NC, the other wells were given 40 μg/mL SiO 2 suspension. e cells were taken out after 4 h in the incubator. 15% DMS was added to DMS + PDTC group and DMS group and the mixture was rested for 12 h. ereafter, 10 μL of CCK8 test reagent (C0037, Shanghai, China) was added to each well, and then the culture was continued for 2 h. Using an ELISA microplate reader (BioTeK Epoch, USA) to measure the optical density (OD) value at 450 nm.

Flow Cytometry Analysis.
e cell suspension in each well was replaced with 200 μL fresh 1× binding buffer. en, annexin V-FITC staining fluorescent dye (BMS500fi-300, Waltham, MA) was added 5 μL per well for 10 min; 10 μL PI staining was added, 5 min later; 400 μL binding buffer was added, mixed fully and detected immediately. e data were analyzed using Cytoflex flow cytometry (Backman, USA) and Kaluza 2.1 software.

Determination of Cytokines.
e ELISA kits of TNF-α (1102589), IL-1β (1102590), and IL-6 (1099199) were obtained from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Shanghai, China). e enzyme plate was sealed after adding the sample and incubated at 37°C for 30 min, washed 5 times with detergent, and incubated at 37°C for 30 min with the HRP labeled enzyme labeled reagent. e plate was washed 5 times again, and the color reagent was applied 20 min later and the reaction was stopped by adding the termination solution. OD at 450 nm was analyzed.

Western Blot Analysis.
Use the BCA protein assay kit (P0009, Shanghai, China) to detect protein concentration. First, extract the protein: take out the sample to be tested, add RIPA lysis solution to each EP tube containing the tissue to be tested, and place it on crushed ice for 10 minutes; collect the lysis solution and centrifuge for 10 minutes at 4°C and 12000 rpm; take the clear liquid to be tested. Second, protein content determination: use BCA protein quantification kit to determine protein concentration. Dilute 2 μL of the sample to be tested to 16 μL with lysate, then add 2 μL of the diluted protein to the well, add lysate to make up to 20 μL, add BCA working solution, mix well, and incubate at 37°C for 30 minutes. With No. 0 hole as the control, measure the OD value of the sample at the wavelength of 562 nm; draw a standard curve based on the value obtained from the standard and calculate the protein content of the corresponding sample. After electrophoresis, the proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Being sealed in 5% skimmed milk for 1 h at room temperature, the diluted primary antibody was incubated at 4°C overnight. e primary antibodies including p38 mitogen-activated protein kinase (p38 MAPK, 1 : 200, SC-7973, Santa Cruz, CA), nuclear factor kappa B (NF-κB) p65 (1 : 200, SC-514451, Santa Cruz, CA), transforming growth factor-beta1 (TGF-β1, 1 : 1000, GB11179, servicebio, Wuhan), alphasmooth muscle actin (α-SMA, 1 : 1000, GB13044, servicebio, Wuhan), Smad2 (1 : 1000, GB11511, Servicebio, Wuhan), Smad3 (1 : 1000, bsm-52224r, bioss, Beijing), and Smad7 (1 : 200, SC-365846, Santa Cruz, CA). After being washed 3 times with Tris-Buffered Saline Tween-20, the membranes were incubated with secondary antibody (1 : 5000, ab6721, Abcam, Cambridge) at room temperature for 2 h, then washed 3 times again. After mixing A and B reagents of enhanced chemiluminescence luminescent solution (ECL, KF001, Affinity Biosciences, OH), drop them onto the membrane uniformly for 1 minute and then put the membrane into the dark room of the chemiluminescence gel imager. Images were captured with a Gene Gnome Gel Imaging System (Syngene, USA) and Image J Software was used to analyze the gel images. e antibodies are as described above. After the slides of cells were spin-dried, 50-100 μL of membrane breaking working solution was added and incubated 20 min, then it was washed with phosphate-buffered saline for 3 times. e cells were covered with 3% bovine serum albumin, sealed 30 min, and then incubated overnight with primary antibody at 4°C. e next day, sections were incubated with fluorescein isothiocyanate-conjugated secondary antibody for 50 min at room temperature, after dripping of 4′,6-Diamidino-2phenylindole (DAPI, G1012, OH, USA) dye solution and were incubated in the dark for 10 min. Images were photographed by fluorescent microscopy (Nikon Eclipse C1, Japan).

Statistical Analysis.
e data in this experiment are analyzed using SPSS25.0 software. e data are presented as the mean ± SEM. Intragroup comparison: paired T-test is used for normal distribution, and rank sum test is used for nonconforming distribution. Comparison between groups: one-way analysis of variance (One-Way ANOVA method) is used for normal distribution, LSD test is used for uniform variance, Tamhane's T2 test is used for uneven variance, rank sum test is used for nonnormal distribution. P < 0.05 is considered to be a statistically significant difference, P < 0.01 is considered a significant difference.

e Contents of 5 Components of Traditional Chinese Medicine in DMS.
e results of the reference standard compounds and DMS samples were shown in Figure 1, and the contents were shown in Table 3. 5 major components of DMS were verified by comparing individual peak retention time and quantifier ions with the reference standard compounds.
e peak area of amygdalin, baicalin, catalpol, emodin, and paeoniflorin were taken as the vertical coordinate and the concentration as the horizontal coordinate, and the standard curve was obtained by regression with the weighted coefficient (Equal). Table 4, 40 μg/mL SiO 2 significantly decreased the MH-S cell viability compared with normal cells. However, 15% DMS and 4 mM PDTC effectively improved the viability of MH-S cells to different degrees, especially the combination of DMS and PDTC, suggesting that DHZCP reduced the cell damage caused by SiO 2 . It can be seen from the data statistics that compared with the NC group, the cell viability of MC was significantly impaired, and there was a significant difference (P < 0.001). Compared with MC in DMC + PDTC and DMS groups, cell viability was significantly restored (P < 0.001 and P < 0.01). Compared with the MC group, the PDTC group showed a tendency to restore cell viability.

DHZCP Inhibits SiO 2 -Induced Apoptosis in MH-S Cells.
As Figure 2 shows, apoptosis significantly increased under stimulation of SiO 2 , as expected. In the MC group, the total apoptosis rate was 11.56%. Compared with the MC group, the DMS group and PDTC group significantly reduced the apoptosis rate. However, DMS and PDTC combined treatment was more effective, as the total apoptosis rate was only 3.87%. Compared with PDTC group, the total apoptosis rate of the DMS group was lower than the PDTC group, which indicated that DMS was better than PDTC in reducing apoptosis. Table 5, compared with normal MH-S cells, the levels of TNF-α, IL-1β, and IL-6 in the MC group were significantly increased, and TNF-α and IL-1β were significantly increased (P < 0.001), and there were significant differences. After DMS and PDTC treatment, the contents of TNF-α, IL-1β, and IL-6 in the cell supernatant were significantly reduced. Among them, TNFα was significantly different in the DMS + PDTC group, DMS group, PDTC group, and the model group (P < 0.001). Compared with the model group, the content of IL-1β is different in the DMS group (P < 0.01), and the difference is significant (P < 0.001) in the DMS + PDTC group and the PDTC group (P < 0.001), and the content of IL-6 is present compared with the model group; it showed a downward trend, but it was not statistically significant. Figure 3, compared with normal cells, the protein expression of p38 MAPK, NF-κB p65, TGF-β1, α-SMA, Smad2, and Smad3 in MH-S cells induced by SiO 2 increased significantly. Among them, the increase of p38 MAPK, NF-κB p65, p-p65, TGF-β1, α-SMA, Smad2 was significantly different from the model group (P < 0.001); the increase of p-p38 and Smad3 was also statistically significant (P < 0.01, P < 0.05). On the contrary, the model Smad7 of the group was significantly lower than that of the normal group (P < 0.001). However, after DMS treatment, the expression of these proteins significantly decreased. e trend of Smad7        Evidence-Based Complementary and Alternative Medicine protein expression was the opposite. Interestingly, PDTC, an inhibitor of NF-κB, was used to interfere with SiO 2 -induced MH-S cells. In addition to the protein of p38 MAPK, p-p38, PDTC also significantly decreased the expression of NF-κB p65, p-p65, TGF-β1, α-SMA, Smad2, Smad3 proteins and increased the expression of Smad7 protein. Moreover, the combined effect of PDTC and DMS was better than that of the two being used separately, except for smad3, there are significant differences between other indicators and the model group (P < 0.001). And this regulatory effect of DHZCP in SiO 2 -induced MH-S cells was further confirmed by immunofluorescence staining, as indicated in Figure 4.

Discussion
e aim of this study was to explore the preventive and therapeutic effects of DHZCP on silicosis and the mechanisms. Persistent inflammatory stimulation is an important cause of fibrosis [14]. e inhaled SiO 2 particles were phagocytized by alveolar macrophages because when the body experiences an inflammatory response, it activates a natural immune response, and the activated macrophages release inflammatory cytokines, which could attract more monocyte or macrophage apoptosis. As the SiO 2 clearance function was impaired, lung tissues gradually develop persistent pulmonary inflammation [7], alveolar macrophages, fibroblasts, and endothelial cells initiate abnormal repair patterns, leading to the deposition of the extracellular matrix, and ultimately to the formation of irreversible abnormal remodeling and fibrosis of lung tissue [15]. According to the Chinese Pharmacopoeia, we determined the contents of Amygdalin, Baicalin, Catalpol, Emodin, and Paeoniflorin in DHZCP medicated serum and then used them in the experiment to ensure the validity of the experiment. In this study, we found that DHZCP could improve vitality and significantly decrease the apoptosis rate of SiO 2 -induced MH-S cells. It was also found that DHZCP could reduce the levels of cytokines (TNF-α, IL-1β, IL-6) in cell supernatant significantly. In addition, PDTC can not only affect the DNA binding activity but also directly suppress NF-κB and depress the nuclear translocation of NF-κB, thereby decreasing the two subunits p50 and p65 of NF-κB transferred to the nucleus to play an inhibitory role. erefore, the expression of TNF-α, IL-6, and IL-1β genes effectively reduces the release of neutral hydrolase, peroxidase, and acid hydrolase, which are destructive to tissues from the inflammatory sites,   Quantitation of p38 MAPK, p-p38, NF-κB p65, p-p65, TGF-β1, α-SMA, Smad2, Smad3, and Smad7 in MH-S. e experiment was repeated three times. n � 3. * P < 0.05 and * * * P < 0.001 VS the NC group, # P < 0.05, ## P < 0.01 , and ### P < 0.001 VS the MC group. 8 Evidence-Based Complementary and Alternative Medicine thereby reducing the inflammatory response. Blocking NF-κB activation is an effective way to control the overall pathway upstream of an inflammatory reaction and prevent organ dysfunction. p38 MAPK, the upstream gene of NF-κB, could be activated by inflammation and stress reaction [16][17][18]. In our study, the expression of NF-κB p65 and p-p65 in cells stimulated by SiO 2 was significantly upregulated; however, they were significantly downregulated in pretreated PDTC cells stimulated by SiO 2 . In addition, the expression of p38 MAPK and p-p38 in the upstream did not change significantly, while the TGF-β1, Smad2, Smad3 in the downstream reduced accordingly. It shows that PDTC pretreatment could downregulate the expression of NF-κB p65 and nuclear expression level of  Evidence-Based Complementary and Alternative Medicine p-p65 by reducing NF-κB activation so that the terminals of the pathway could not receive the stimulation signal from SiO 2 , thus inhibiting the release of the TNF-α, IL-1β, IL-6, and the development of inflammation. It also indicated that PDTC had no significant effect on the upstream p38 MAPK. When DHZCP was applied to MH-S cells activated by SiO 2 , in addition to declining the NF-κB p65 and p-p65 to block the TGF-β1/Smad signaling pathway, it also significantly downregulated the expression of p38 MAPK and p-p38. It was suggested that DHZCP could inhibit inflammation and TGF-β1/Smad signaling pathway by blocking NF-κB activation; it also could inhibit p38 MAPK upstream of NF-κB. DHZCP could control the inflammatory reaction from the upstream with multiple targets, which is more effective than the single inhibition of NF-κB by PDTC. is was also in line with the law of traditional Chinese medicine multitarget treatment of diseases. As mentioned above, DHZCP could decrease the levels of inflammatory cytokines in MH-S cells induced by SiO 2 . ese results indicated that DHZCP could effectively reduce inflammation in the early stage of silicosis and eliminate or alleviate the continuous inflammatory stimulation of SiO 2 .
NF-κB, an upstream regulatory gene for TGF-β1 expression, when NF-κB is activated, can induce the expression of TGF-β1 [19]. In the case of inflammation, NF-κB is phosphorylated and transferred to the nucleus to become a nuclear transcription factor, and it induces the expression of TGF-β1 [19]. It can be seen that in the model group, with the increase of p-p65, the expression of TGF-β1 also has an upward trend. On the contrary, after using DHZCP, NF-κB p65, p-p65, and TGF-β1 showed a downward trend. TGF-β1 is recognized as the most important initiator of pulmonary fibrosis [20][21][22]. TGF-β1 is a major fibrogenic factor that increases the synthesis of α-SMA and continuously activates fibroblasts into myofibroblasts with contractile function and promotes the accumulation of ECM proteins in the lung interstitium and alveolus [23]. Smad protein is downstream signal transduction and a regulatory molecule that mediates the signal transfer from cell membrane to the nucleus [24]. At present, the protein levels of TGF-β1, α-SMA, Smad2, and Smad3 in MH-S cells stimulated by SiO 2 were significantly increased, while Smad7 was decreased, indicating that SiO 2 stimulation successfully activated TGF-β1 to initiate fibrosis and activated the downstream Smad signal. After DHZCP treatment, TGF-β1, α-SMA, Smad2, and Smad3 were significantly decreased, while Smad7 was increased.
ese results indicated that DHZCP downregulated TGF-β1 and regulated the downstream Smad signaling pathway, effectively inhibiting the initiation of fibrosis. erefore, the protective effect of DHZCP on SiO 2 induced MH-S cells is related to downregulating TGF-β1 and p38 MAPK/TGF-β1/Smad pathway.

Conclusions
In conclusion, we provide evidence that DHZCP could reduce the inflammatory response of MH-S cells induced by SiO 2 , eliminate the sustained inflammatory stimulation, aand effectively inhibit the initiation of fibrosis. DHZCP's strong protective effects in the SiO 2 -induced silicosis fibrosis model are related to the p38 MAPK/TGF-β1/Smad pathway.

Data Availability
e datasets used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Li-Juan Wu and Xiao-Yan He contributed equally to this work. LJ, HX, WL, and CD were the guarantor of the integrity of the entire study and contributed to the study concepts and design; HX, WL, and ZY performed experiments. HX, WL, and LJ analyzed and interpreted the data. WW and LJ contributed to the literature search and data collection; HX, WL, and WW contributed to the manuscript preparation and revision, and CD reviewed the manuscript. All the authors discussed and approved the final version.