Effect of TAK242 on MCP-1 and TGF-β in COPD Rats

Objective: To investigate the mechanism of MCP-1 and TGF-β regulation by TAK242 in COPD rats. Methods: Thirty-six SD rats were randomly divided into normal, COPD control, and TAK242 groups. The normal group was freely fed, and the other groups used the method of fumigation plus lipopolysaccharide tracheal drip to establish an experimental animal model of COPD. After successful modeling, each experimental group received 0.9% NaCl solution and corresponding drugs by intraperitoneal injection for 7 d. After drug administration, lung function was examined; pathological changes in lung tissue were observed by light microscopy with hematoxylin-eosin staining; mRNA expression of MCP-1 and TGF-β was detected by q-PCR; and protein expression of MCP-1 and TGF-β in lung tissue was detected by Western blot and IHC, TGF-β protein expression in rat lung tissue. Results: Compared with the normal group, rats in the COPD control group showed signs and symptoms of COPD, decreased lung function, and increased expression of MCP-1 and TGF-β. The TAK242 group showed decreased expression of MCP-1 and TGF-β compared to the COPD control group. Conclusion: MCP-1, and TGF-β played a crucial role in the early stage of COPD (cid:976)ibrosis. TAK242 could ameliorate airway in(cid:976)lammation and inhibit the progression of COPD lung (cid:976)ibrosis in pre-existing rats in COPD model rats.


Introduction
COPD due to long-term inhalation of smoke and other pollutants, caused by in lammatory reactions, lung infections, or deposition of interstitial immune complexes in the lung and other factors, inducing the production of ibrotic cytokines, which is the main cause of the formation of chronic pulmonary ibrosis [1][2][3].Pulmonary ibrosis is also a major challenge in the diagnosis and treatment of lung disease.It is therefore important to delay the progression of pulmonary ibrosis and to ind effective treatments.As the pathogenesis of pulmonary ibrosis is better understood, there is increasing evidence that in lammation is also a recognized factor in the pathogenesis of pulmonary ibrosis [4].In lammatory responses such as immune in lammatory cells and cytokines play an important role in the development of pulmonary

Effect of TAK242 on MCP-1 and TGF-β in COPD Rats
Ruicheng Deng 1# , Mingyu Duan 2# , Xiaoyong Ma 3 , Juanxia Chen 4 , Huifang Zhang 4 , Meifang Liu 4 , Jian Chen 2 and Lijun Chen 4 * ibrosis.The in lammatory response in pulmonary ibrosis is caused by the increased expression of monocytes and macrophages in lung tissue; MCP-1 and TGF-β are two important causes leading to the in iltration of monocytes and macrophages into lung tissue [5,6].MCP-1 is one of the major in lammatory factors involved in lung injury and plays a key role in exacerbating the progression of COPD pulmonary ibrosis.As a member of the in lammatory chemokine family, MCP-1 mediates the in iltration of a wide range of in lammatory cells in COPD lung ibrosis, with the number of monocytes and macrophages being directly proportional to the degree of interstitial ibrosis [7].TGF-β is the major cytokine involved in the pathogenesis of pulmonary ibrosis in COPD.The in lammatory environment enhances oxidative stress-induced TGF-β expression [8], while TGF-β promotes cellular hypertrophy, ECM accumulation, and EMT.Among https://doi.org/10.29328/journal.jro.1001060 the many regulators of EMT, TGF-β is considered an important factor in pulmonary ibrosis [9].Although many studies have elucidated the formation mechanism of pulmonary ibrosis, the intrinsic in luence of in lammatory mediators in the disease remains to be further investigated and con irmed.In lammation inhibition has the characteristics of multipathway and multi-target, which has certain advantages in the prevention and treatment of pulmonary ibrosis, and plays an important role in the treatment of pulmonary ibrosis, with comprehensive therapeutic advantages in anti-in lammatory, antioxidant, and delaying pulmonary ibrosis [10][11][12].Among them, TAK242 belongs to the toll-like receptor inhibitors, which affect the NF-kB signaling pathway, which belongs to the typical in lammatory pathway, and inhibition of which has the functions of reducing in lammation, improving the degree of ibrosis to regulate immunity and antioxidant [13,14], etc.In this study, based on previous studies and the ef icacy of known drugs, we established a COPD model and observed the expression of pulmonary ibrosis factors MCP-1 and TGF-β in lung tissues to explore the therapeutic role and mechanism of TAK242 in the early stage of pulmonary ibrosis in COPD.

Experimental animals
Basic information: Thirty-six healthy SPF-grade male SD rats with an average body mass of (180±10) g were provided by the Experimental Animal Centre of Ningxia Medical University (SYXK Ning 2020-0001), randomly divided into the COPD group and the normal group, with 13 rats in the normal group and 23 rats in the COPD group, all housed in a clean SPF grade environment.At the time of modeling, 3 rats each from the normal and COPD groups were randomly selected to verify the success of the model, and then the COPD group was randomly divided into a COPD control group (10 rats) and a TAK242 group (10 rats).

Methods
COPD rat model preparation: Lipopolysaccharide (200 μg/time) was injected into the trachea of rats in the COPD group on days 1, 14, 28 and 42; the rats were placed in a home-made poisoned box for passive smoking on days 2-13, 15-27 and 29-41 (2 times a day, each time for 60 min, with an interval of > 8 h between the two times, and each time for 10 cigarettes); the modeling was performed for a total of 42 d [15][16][17].The rats in the normal group received no special treatments.At the end of the modeling, 3 rats from each of the COPD and normal groups were randomly selected and the air low limitation of the COPD group rats was assessed using a small animal spirometer; The rats were anesthetized with sodium pentobarbital, and then the chest was opened after execution under anesthesia, and the lung tissues were collected for HE staining, which veri ied that the lung function and histopathological changes of the COPD group rats were consistent with the characteristics of COPD, and con irmed that the COPD rat model was successfully established.The rat model of COPD was successfully established.Marginal notes: Take 50 μL of 102 mg/ml of clari ied DMSO stock solution and add it to 400 μL of PEG300, mix well to make it clari ied; add 50 μL of Tween80 to the above system, mix well to make it clari ied; and then continue to add 500 μL of ddH2O to ix the volume to 1 mL.

Normal group: free feeding, no intervention.
COPD control group: COPD rats were injected intraperitoneally with the same volume of saline once a day for 7 consecutive days.
TAK242 group: TAK242 was administered to COPD rats by intraperitoneal injection at 6 mg/kg once a day for 7 consecutive days.

Pulmonary function measurements in rats:
Rats were anesthetized with 2% pentobarbital (50 mg/kg) by intraperitoneal injection, the trachea was incised in the middle of the neck, and the tracheal tube was inserted and placed in the body box.Total lung capacity (TLC), 200 ms rate (FEV200/FVC), and 100 ms rate (FEV100/FVC) were measured using a small animal spirometer.https://doi.org/10.29328/journal.jro.1001060

Rat lung tissue sampling and pathological observation:
Rats were killed by air embolization, and the left lung was removed from the open chest and immersed in 14% paraformaldehyde ixative for 24 h.The lung was rinsed with phosphate buffer solution and then serially sectioned by conventional paraf in embedding and stained with HE.The upper lobe of the right lung of rats was placed in a freezing tube, frozen in liquid nitrogen and then stored in a refrigerator at -80 ℃.
Determination of MCP-1 and TGF-β mRNA in rat lung tissues: Total RNA was extracted from frozen right lung tissues according to the instructions of the TIANGEN RNA Kit, and the OD260/280 was measured by UV spectrophotometer at 1.8-2.0.cDNA was synthesized according to the instructions of the Reverse Transcription Kit, and the mRNA concentration was determined using a qPCR kit.The reaction system consisted of 20 μL, 10 μL TB Green Premix, 0.8 μL forward primer, 0.8 μL reverse primer, 2 μL DNA template, and 6.4 μL of sterilized water, and the reaction conditions were as follows: pre-denaturation at 95 ℃ for 30 s, and cycling for 40 times according to the following procedure: denaturation at 95 ℃ for 5 s, annealing at 57 ℃ for 30 s, and extension at 72 ℃ for 1 min.The relative expression of MCP-1 and TGF-β was determined by the 2-ΔΔCt method using the release curve of the instrument and ACTB as an internal reference.

Western blot detection of MCP-1, TGF-β protein expression:
The rats in normal, COPD, and TAK242 groups were killed under anesthesia, then the chest was opened, the right lung tissues were quickly taken out and put into liquid nitrogen, the rat lung tissues were taken out and ground, and the protein immunoblotting method was applied for protein quanti ication of rat lung tissues for MCP-1 and TGF-β.Total tissue protein was extracted according to the instructions of the kit, and protein quanti ication was performed by the BCA method.Take 40μg of protein and add the sample buffer to denature for use.Preparation of SDS-PAGE gel, concentrated gel 100V voltage electrophoresis 40min, separated gel 120V voltage electrophoresis 1h, 200mA constant current wet rotation 80min, the protein will be transferred to the PVDF membrane, Li Chun red staining to observe the bands, TBST wash the membrane, 5% skimmed milk TBST closure of 1. 5h, add a primary antibody incubated at room temperature for 2h, TBST wash the membrane, add a secondary antibody reaction of 1h, TBST wash the membrane, ECL luminous reagent luminescence, ilm exposure development and ixation.TBST washed membrane, ECL luminous reagent luminescence, ilm exposure, development, and ixation.The images were captured and the results were scanned for absorbance using gel image analysis software, and the ratio of absorbance of MCP-1 and TGF-β protein bands to GADPH was used as the amount of MCP-1 and TGF-β protein expression.The differences in MCP-1 and TGF-β protein expression in rat lung tissues of the two groups were compared.

Immunohistochemical staining:
After paraf in embedding and sectioning, tissue sections were routinely deparaf inized and dehydrated, 3% hydrogen peroxide (H202) deionized water was closed to peroxidase, the reaction was carried out for 10 minutes, and rinsed in distilled water for 5 minutes × 3 times.In citrate buffer, microwave heat repair, boiling twice, PBS rinse, 5 min × 3 times.Reagent A (blocking solution, blue liquid) was added dropwise, incubated for 20 min at room temperature, decanted, do not wash.Add the corresponding primary antibody dropwise, overnight at 4 °C, 30 min at room temperature, and rinse in PBS, 5 min × 3 times.Drops of reagent B (the corresponding secondary antibody working solution, yellow liquid), room temperature incubation for 15 minutes, PBS rinse, 5 minutes × 3 times, drops of reagent C (horseradish enzymelabeled streptavidin working solution, orange liquid), room temperature incubation for 20 minutes, PBS rinse, 5 minutes × 3 times.DAB color development (microscopic observation of the color of the color to terminate the color development), the tap water suf iciently washed, and hematoxylin restaining 30 seconds.Dewatering, transparency, sealing.The positive cells were determined by the presence of brownish-yellow granular deposits in the cytoplasm or nucleus.Comparison of the semiquantitative and localized expression of MCP-1 and TGF-β proteins in the lung tissues of four groups of rats.

Statistical methods
The experimental data were expressed by the mean standard deviation (x̄ ± S), they were processed by SPSS statistical software.One-way ANOVA was used to compare multiple samples.With a difference of p < 0.05 being statistically signi icant.

Signifi cant eff ect on lung function in COPD rats
In order to verify the effect of COPD on the lung function of rats, we tested FEV100/FVC and FEV200/FVC parameters, and the results showed that there was a signi icant difference in FEV100/FVC and FEV200/FVC between rats in the control group and rats in the COPD group (p < 0.05), but there was no difference in the improvement of lung function of rats in the TAK242 group (p > 0.05) (Figure 1).

Diff erent interventions have signifi cant eff ects on the general condition and histopathological changes in the lungs of COPD rats
The rats in the normal group had normal activity and diet throughout the modeling period, shiny hair, good mood, and normal respiration; HE of lung tissue showed that the bronchial walls were regular without in lammatory cell in iltration and the alveolar structure was normal without pathological expansion (Figure 2A).Compared with the normal group, rats in the COPD group gradually reduced their activities and food consumption with the prolongation of the exposure time to LPS+ smoke, their fur became dull and pale yellow, and their mood was depressed, accompanied https://doi.org/10.29328/journal.jro.1001060expression of MCP-1 protein in the lung tissue of rats in the COPD control group was signi icantly higher than that in the normal group; the expression of TGF-β protein in the lung tissue of rats in the COPD group was signi icantly higher than that in the control group; (p < 0.05, Figure 3).

Changes in MCP-1 and TGF-β protein levels in lung tissues of diff erent intervention groups
We used WB to semiquantitatively detect the results of the above IHC experiments, and the results showed that the expression trend of MCP-1 and TGF-β was consistent with the results of the IHC experiments, and the statistical analysis of the grey value suggested that the expression of MCP-1 and TGF-β was high in the COPD group, and the expression level of the normal group was low (p < 0.05, Figure 4).

Diff erent interventions regulate the relative expression of MCP-1 and TGF-β mRNA in rat lung tissue
There were differences in the levels of MCP-1 and TGF-β in the lung tissues of rats in each group.The levels of MCP-1 and TGF-β in the lung tissues of rats in the COPD control group were signi icantly higher than those in the normal group (p < 0.05).(Figure 5).

Discussion
Pulmonary ibrosis is one of the complications of COPD [1,18], with mild clinical manifestations in the early stage and the following pathological features: early lesions are mainly con ined to the alveolar wall, the alveolar lumen is illed with luid and cellular exudates, and the structure of the alveoli is blurred and disappears; then there is the destruction of ibrous tissue, disorganization of the lung structure, and the gradual development of interstitial ibrosis.Numerous studies have shown the presence of in lammatory cell in iltration, elevated cytokines, and increased levels of in lammatory mediators in pulmonary ibrosis [19] and in the early stage of pulmonary ibrosis, monocyte/macrophage in iltration is present in all lung tissues [20].In iltrating monocytes/macrophages destroy lung structures by releasing cytokines, in lammatory mediators, and oxygen free radicals, accelerating the process of lung tissue sclerosis.In this study, after the success of the COPD model, MCP-1 and TGF-β were found to be elevated  by congestion of the eyeballs and the appearance of nasal secretions; HE of lung tissues showed mucus secretion and in lammatory cell in iltration of bronchial tubular lumen, submucosal glandular hyperplasia, disorganization of alveolar structure, and enlargement of alveolar lumen coalescing into pulmonary pustules (Figure 2B).Compared with the COPD control group, the TAK242 group showed a signi icant reduction in bronchiolar luminal mucus and in lammatory cell in iltration, and the rats' activity and diet improved signi icantly, with bright fur color and good mood.The HE of the lung tissue showed signi icantly less mucus secretion and in lammatory cell in iltration in the bronchial lumen, and the changes in the alveoli and glands were not obvious (Figure 2C).

Diff erent interventions regulate the expression of MCP-1 and TGF-β in lung tissue
We used IHC to detect MCP-1 and TGF-β protein expression in rat lung tissues, and the results showed that the expression of each protein index was high in the COPD group and lowest in the normal group.Among them, the https://doi.org/10.29328/journal.jro.1001060 in rat lung tissue, but the HE of lung tissue did not show signs of ibrosis, but the elevation of MCP-1 and TGF-β was positively proportional to chronic lung ibrosis, con irming that there is an obvious causal relationship between COPD and lung ibrosis.Our use of TAK242 to inhibit toll-like receptors resulted in a signi icant decrease in MCP-1 and TGF-β and had the function of delaying lung ibrosis.In our study, we found that TAK242 did not signi icantly improve lung function in rats, suggesting that inhibition of this in lammatory pathway was not able to reverse alveolar damage.Other studies have shown that in lammation is a key factor in the pathogenesis of lung ibrosis [21].Although COPD is a recurrent in lammatory process, many cytokines are involved in the in lammatory response, such as interleukin-6 (IL-6), TGF-β, MCP-1, etc [7,22,23].They play different roles and form a complex cytokine network.Among them, MCP-1 and TGF-β are closely associated with the progression of ibrosis.MCP-1, as a member of the in lammatory chemokine superfamily, can be released by a wide range of cells and is low expressed under physiological conditions.In contrast, under pathological conditions such as hyperglycemia and oxidized lipoproteins, thylakoid and endothelial cells can stimulate MCP-1 gene expression.On the one hand, high levels of MCP-1 an autocrine or paracrine stimulate the expression of IL-1 and TGF-β produced by alveolar cells and exacerbate endothelial cell injury.On the other hand, alveolar cells can be directly activated to produce myo ibrillar proteins, leading to interstitial ibrosis.Activated macrophages can release reactive oxygen radicals, in lammatory mediators, and growth factors that promote the increase and deposition of ECM (extracellular matrix) [24], ultimately leading to the development of pulmonary arterial sclerosis.Evidence suggests that MCP-1 is a key factor in the early in lammatory process of atherosclerosis and also plays a central role in renal pathology, promoting the development of renal ibrosis and the progression of diabetic nephropathy [25].Transforming growth factor-β, which is predominantly distributed in the lung, is a central factor in the complex cytokine network associated with the pathogenesis of pulmonary ibrosis and regulates tissue damage and repair under normal conditions.Overproduction of TGF-β in patients with pulmonary ibrosis is associated with chronic ibrosis.In addition, TGF-β expression is associated with oxidative stress, which is increased by prolonged hyperglycemia [26].TGF-β mediates the TGF-β/ Smad signaling pathway, which promotes ECM aggregation and decreases the activity of enzymes that degrade ECM, ultimately leading to interstitial lung ibrosis.In addition, TGF-β induces autophagy of renal tubular epithelial cells and promotes apoptosis of renal tubular epithelial cells, leading to tubular injury.Meanwhile, TGF-β is associated with the formation of autophagic proteins, leading to pulmonary capillary ibrosis [27].High expression of TGF-β upregulates and enhances the transcription of MCP-1 through the nuclear transcription factor-κB (NF-κB) signaling pathway.Meanwhile, high expression of MCP-1 can activate the p38-MAPK signaling pathway via in lammatory response factors released by activated monocytes/macrophages to produce TGF-β, which promotes ibrosis and leads to lung lesions [28].Studies have shown that when recombinant TGF-β is present in organisms, MCP-1 levels increase, while macrophages synergizing with MCP-1 can in turn stimulate the production of TGF-β and promote the deposition of ECM, ultimately leading to interstitial lung ibrosis [29].Thus, TGF-β and MCP-1 directly or indirectly interact to form a vicious circle, exacerbating the in lammatory response and promoting the progression of pulmonary ibrosis.Therefore, MCP-1 and TGF-β play a crucial role in the development of ibrosis, suggesting that MCP-1 and TGF-β can be used as biomarkers to assess the development of pulmonary ibrosis.TAK242 can effectively inhibit the production of TGF-β and MCP-1, after which it can effectively reduce the local aggregation of monocytes/macrophages, reduce the deposition of ECM, and delay the progression of pulmonary ibrosis.TAK242 and its activated components have important biological effects such as anti-in lammatory, immunomodulatory, anti-tumor, and antioxidant effects [30].This experiment showed that the use of TAK242 over a period of time could alleviate some pathological changes in the early stage of pulmonary ibrosis, such as basement membrane thickening and ECM accumulation [13].Lung ibrosis can occur in the rat COPD model, and EMT is an important mechanism for the development and progression of interstitial lung ibrosis [31].TAK242 was found to be effective in reducing the in lammatory response in the lung and ameliorating the in lammatory situation in rats in vivo.In terms of mechanism study, the expression of MCP-1 and TGF-β was signi icantly upregulated in the COPD control group, and TAK242 could inhibit the expression of MCP-1 and TGF-β in lung tissues.Our results suggest that TAK242 plays a role in the prevention of COPD lung ibrosis by inhibiting, attenuating, and treating MCP-1 and TGF-β, key factors in in lammation and ibrosis.In addition, we found that TAK242 reduced in lammatory cell in iltration in HE-stained sections.In recent years, a growing body of new evidence has demonstrated that TAK242 has broad therapeutic ef icacy and comprehensive therapeutic value with fewer side effects.Given these advantages, TAK242 is a promising chemical agent.There are some possible limitations to this study.First, this experiment was limited to animals only, which may lead to bias in clinical trials.TAK242 has a variety of effects such as antioxidative stress, anti-in lammation, and immunomodulation, and only a small part of anti-in lammatory cytokines and in lammatory effects were shown in this study.Therefore, the mechanism of the protective effect of TAK242 on COPD lung ibrosis needs further investigation.

Conclusion
The present study demonstrated that TAK242 has a promising protective effect against COPD-induced lung ibrosis.These bene icial effects were closely associated with the downregulation of MCP-1 and TGF-β expression, which is important for the development of new strategies for the prevention and treatment of pulmonary ibrosis in COPD.

Compliance statement
This research has been conducted in accordance with

Figure 2 :
Figure 2: The general condition of the rats and histopathological changes in the lungs (A).HE staining of lung tissue in the control group.(B) HE staining of lung tissue of the COPD group.(C) HE staining of lung tissue in the TAK242 group.

Figure 3 :
Figure 3: Expression of MCP-1 and TGF-β in lung tissue from different groups.

Figure 5 :
Figure 5: Relative expression of MCP-1 and TGF-β mRNA in plasma of rats regulated by different interventions.(A) MCP-1 mRNA expression level in lung tissue of each group.(B) TGF-β mRNA expression levels in lung tissues of each group.