CN110208526A - CoQ10Application in anti-silicotic fibrosis - Google Patents

Abstract

The invention discloses the application of CoQ ₁₀ in anti-silicosis fibrosis, which is characterized in that it comprises the following steps: (1) setting a control group: selecting 24 male C57 mice of SPF grade 6 weeks old, with a body weight of 20 ± 2g, and first adapting to After sexual feeding for one week, they were randomly divided into control group, model group, and treatment group; the model group and treatment group were instilled with 0.1mL SiO ₂ suspension into the trachea once to construct the mouse silicosis model, and the control group was instilled with the same volume of normal saline ; 48 hours after operation, the treatment group was given 0.1mL CoQ ₁₀ per day, orally, and the control group and model group ate independently. The present invention has the following advantages: CoQ ₁₀ reduces _SiO2 -induced silicosis fibrosis, CoQ ₁₀ can down-regulate the expression of collagen, hydroxyproline and α-SMA, and its mechanism of action may be feedback regulation of endogenous HMGCR expression, affecting oxidation The respiratory chain provides a new perspective for the study of the mechanism of subsequent silicosis treatment.

Description

Application of CoQ10 in anti-silicosis fibrosis

technical field

The invention relates to the technical field of medical engineering, in particular to an application of CoQ ₁₀ in anti-silicosis fibrosis.

Background technique

At present, more than 850,000 cases of pneumoconiosis have been reported in my country, of which silicosis accounts for more than 50%. It is characterized by the inhalation of SiO ₂ particles into the lungs, stimulating lung tissue to initiate self-repair mechanisms, and activating fibroblasts to produce collagen fibers. However, when the lung tissue is repeatedly or overstimulated, fibroblasts proliferate abnormally and secrete a large number of collagen fibers, resulting in excessive deposition of collagen in the lung interstitium, and fibrosis of the lung interstitium. Clinical treatment mainly focuses on bronchoalveolar lavage combined with anti-fibrosis and symptomatic treatment of complications. At the same time, comprehensive treatment strategies such as oxygen therapy and psychological counseling are given, but there is no specific treatment plan. Therefore, whether it can be excavated from the treasure trove of existing drugs New drugs that are safer, more convenient, and have multiple advantages in prevention and treatment are of great significance for alleviating patients' symptoms and reducing social burdens.

Because of its anti-oxidation, anti-fibrosis, free radical scavenging, vasodilation, and anti-inflammation effects, CoQ ₁₀ is widely used to reduce blood viscosity and improve coronary artery revascularization and ischemia and reperfusion injury. Studies have found that CoQ ₁₀ can inhibit liver fibrosis by inhibiting the expression of TGFβ-1. At the same time, CoQ ₁₀ can also relieve arginine-induced pancreatic fibrosis and reduce collagen deposition in pancreatic tissue.

Contents of the invention

In order to solve the above-mentioned key problems, the technical solution provided by the present invention is the application of CoQ ₁₀ in anti-silicosis fibrosis, which is characterized in that it comprises the following steps:

(1) Set up reference group: select 24 male C57 mice of SPF grade 6 weeks old, weighing 20±2g, first adaptively feed them for one week, and then randomly divide them into control group, model group, and treatment group; 0.1mL SiO ₂ suspension was instilled once to construct the mouse silicosis model, and the control group was instilled with the same volume of normal saline in the same way; 48h after operation, the treatment group was given 0.1mL CoQ ₁₀ (0.2mg/mL) per day. Stomach, the control group and the model group ate independently;

(2) Preparation of silicosis model: after the mice were anesthetized with isoflurane, the trachea was exposed, and 0.1 mL of normal saline was instilled in the control group, and 0.1 mL of SiO ₂ suspension was instilled in the model group and the treatment group respectively, and then the skin was sutured , to observe the survival status of the mice; 48h after the operation, each mouse in the treatment group was intragastrically given 0.1mL CoQ ₁₀ every day; 60 days later, the mice were anesthetized and killed with 0.3mL0.3% urethane intraperitoneally, and the middle lobe of the right lung was taken. Rinse with normal saline and fix with 4% paraformaldehyde, and the rest of the lung tissue is stored in a -80°C refrigerator;

(3) HE staining and Sirius red staining: mouse lung tissue was fixed with 4% paraformaldehyde in the right middle lobe of the lung for 48 hours, dehydrated, embedded, sliced, sliced at a thickness of 8 μm, baked at 56°C for 6 hours, and then performed HE Dyeing and Sirius red staining;

(4) Determination of hydroxyproline: Accurately weigh 50 mg of frozen mouse lung tissue with a balance, cut it into pieces on ice, add 1 mL of hydrolyzate, and fully hydrolyze in a water bath at 96°C for 20 min. After cooling in running water, adjust each When the pH of the tube is 6.0-6.8, add double distilled water to 10mL and mix well, add appropriate amount of activated carbon, centrifuge at 3500rpm for 10min, take 1mL of supernatant for detection; For No. 2 and No. 3 reagents, after mixing, put them in a water bath at 60°C for 15 minutes, after cooling, centrifuge at 3500 rpm for 10 minutes, take 200 μL of the supernatant in a 96-well plate, use a microplate reader to detect the absorbance value of each tube at a wavelength of 550 nm, and calculate The content of hydroxyproline in lung tissue of mice in each group was statistically analyzed;

(5) Real-time fluorescent quantitative PCR reaction: Weigh the lung tissues of mice in each group, cut them into pieces on ice, add RZ lysate, homogenate, extract the total mRNA of the lung tissues of mice in each group, and measure the total mRNA of the samples with a micro-spectrophotometer. The concentration and purity of RNA; the extracted mRNA was reverse-transcribed into cDNA, the concentration of the cDNA product was 0.5 μg/10 μL system, the reverse transcription conditions were 37°C, 15min, 85°C, 5s; then real-time fluorescent quantitative PCR reaction was performed to detect the concentration of each group Differences in the expression of α-SMA and HMGCR in the mouse lung tissue at the transcriptional level, data processing using 2 ^{- △ △ Ct} for relative quantitative analysis of the target gene;

(6) Immunohistochemistry: Paraffin sections of mouse lung tissue in each group were dewaxed with xylene, twice for 10 minutes each time, gradient alcohol dehydration: with 100% alcohol, twice for 5 minutes each time, with 95% alcohol, 85% alcohol Alcohol, 75% alcohol, 50% alcohol, once each, 5min; heat repair with sodium citrate (PH=6) antigen for 15min, then block with goat serum for 10min, incubate α-SMA/HMGCR primary antibody overnight, HRP The labeled goat anti-rabbit/anti-mouse secondary antibody was incubated for 30 minutes, developed with DAB for 6 minutes, stained with hematoxylin for 20 seconds, rinsed with running water for 2 minutes, dehydrated, transparent, and sealed. Take pictures with a 10-fold objective lens under an ordinary light microscope to observe α-SMA in each group And the expression of HMGCR protein, the positive area was counted by Image-Pro Plus6.0, and the result was quantified by the ratio of the IOD value of the positive area to the area of the picture;

(7) Statistics: SPSS20.0 software was used for statistical analysis, and the data of each group were expressed as mean ± standard deviation (x ± s), and the mean comparison among multiple groups was carried out by one-way analysis of variance, and the pairwise comparison between groups was carried out by LSD method, the test level is α = 0.05.

Preferably, the feeding conditions of the adaptive feeding are 12 hours of light, 12 hours of darkness, temperature (23±1)° C., relative humidity of 50% to 65%, and independent drinking and eating.

Preferably, the concentration of the SiO ₂ suspension is 50 mg/mL.

Preferably, the concentration of the CoQ ₁₀ is 0.2 mg/mL.

Preferably, the HE staining steps are: (1) dewaxing: paraffin sections are dewaxed with xylene, twice for 10 minutes each; (2) gradient alcohol dehydration, using 100% alcohol, twice for 5 minutes, using 95 % alcohol, 85% alcohol, 75% alcohol, 50% alcohol, each time for 5 minutes each time; (3) Distilled water washing for 3 minutes; (4) Hematoxylin staining for 6 minutes; (5) Distilled water washing for 10 seconds; (6) Hydrochloric acid ethanol differentiation for 5 seconds (7) Rinse with distilled water for 3 minutes; (8) Eosin staining for 5 minutes; (9) Rinse with distilled water for 5 seconds; (10) 85% alcohol once for 15 seconds, 95% alcohol once for 15 seconds, 100% alcohol twice for 3 minutes each; ( 11) Xylene twice for 3 minutes each time; (12) Neutral gum to seal the slide and examine with an optical microscope.

Preferably, the Sirius Red staining steps are as follows: (1) dewaxing: paraffin sections are dewaxed with xylene, 2 times for 10 min each; (2) Sirius Red staining solution is dripped for 40 min; (3) rinsed with distilled water for 2 min, removed The surface dye solution was routinely dehydrated and transparent; (4) the slide was mounted with neutral gum, and the fibrosis of the mouse lung tissue was observed.

Compared with the prior art, the present invention has the following advantages: CoQ ₁₀ relieves _SiO2 -induced silicosis fibrosis, CoQ ₁₀ can down-regulate the expression of collagen, hydroxyproline and α-SMA, and its mechanism of action may be feedback regulation of endogenous The expression of HMGCR affects the oxidative respiratory chain, which provides a new perspective for the study of the mechanism of subsequent silicosis treatment.

Description of drawings

Fig. 1 is a schematic diagram of HE staining of lung tissue of mice in each group of the application of CoQ ₁₀ of the present invention in anti-silicosis fibrosis.

Fig. 2 is a schematic diagram of Sirius red staining of lung tissue of mice in each group using CoQ ₁₀ of the present invention in anti-silicosis fibrosis.

Fig. 3 is a schematic diagram showing the expression of α-SMA in lung tissue of mice in each group of the application of CoQ ₁₀ of the present invention in anti-silicosis fibrosis.

Fig. 4 is a schematic diagram showing the expression of HMGCR protein in lung tissue of mice in each group of the application of CoQ ₁₀ of the present invention in anti-silicosis fibrosis.

Detailed ways

The reagents involved in the present invention are as follows: SiO ₂ (Sigma Company, batch number 14808-60-7); Coenzyme Q10 (Soleibao Company, batch number: 303-98-0); -7); Sirius red kit was purchased from (LEAGENE, batch number 0402A19); HE staining kit; Hydroxyproline kit (alkaline hydrolysis) (Nanjing Jiancheng Institute of Biological Products, batch number: A030-2); α- SMA rabbit-derived primary antibody (Chengdu Zhengneng Company, catalog number: 347277); HMGCR mouse-derived primary antibody (SANTA Company, catalog number Sc-271595); mouse two-step detection kit (mouse enhanced polymer detection system) ( Zhongshan Jinqiao, Cat. No.: PV-9002); Rabbit Two-step Detection Kit (Rabbit Enhanced Polymer Detection System) (Zhongshan Jinqiao, Cat. No.: PV-9001); Mouse Goat Serum (Zhongshan Jinqiao, ZLI-9022); DAB Chromogenic Kit (Zhongshan Jinqiao, Lot No. K195225A); Reverse Transcription Kit (TaRaKa, Cat. No. RR037A); qPCR Kit (TaRaKa, 3200A).

The present invention will be described in further detail below in conjunction with the accompanying drawings.

In conjunction with the accompanying drawings, (1) Set up a reference group: select 24 male C57 mice of SPF grade 6 weeks old, with a body weight of 20 ± 2g, and first adaptively feed them for one week and then randomly divide them into a control group, a model group, and a treatment group; The treatment group was instilled 0.1mL SiO ₂ suspension into the trachea once to construct the mouse silicosis model, and the control group was instilled with the same volume of normal saline in the same way; the treatment group was given 0.1mL CoQ ₁₀ by intragastric administration every day after 48 hours, and the control group Group and model group ate independently;

(2) Preparation of silicosis model: after the mice were anesthetized with isoflurane, the trachea was exposed, and 0.1 mL of normal saline was instilled in the control group, and 0.1 mL of SiO ₂ suspension was instilled in the model group and the treatment group respectively, and then the skin was sutured , to observe the survival status of the mice; 48h after the operation, each mouse in the treatment group was intragastrically given 0.1mL CoQ ₁₀ every day; 60 days later, the mice were anesthetized and killed with 0.3mL0.3% urethane intraperitoneally, and the middle lobe of the right lung was taken. After flushing with normal saline, it was fixed with 4% paraformaldehyde, and the rest of the lung tissue was stored in a -80°C refrigerator;

(3) HE staining and Sirius red staining: mouse lung tissue was fixed with 4% paraformaldehyde in the right middle lobe of the lung for 48 hours, dehydrated, embedded, sliced, sliced at a thickness of 8 μm, baked at 56°C for 6 hours, and then performed HE Dyeing and Sirius red staining;

(4) Determination of hydroxyproline: Accurately weigh 50 mg of frozen mouse lung tissue with a balance, cut it into pieces on ice, add 1 mL of hydrolyzate, and fully hydrolyze in a water bath at 96°C for 20 min. After cooling in running water, adjust each When the pH of the tube is around 6.0-6.8, add double distilled water to 10 mL and mix well, add an appropriate amount of activated carbon and centrifuge at 3500 rpm for 10 min, and take 1 mL of the supernatant for detection. Set up blank tubes, standard tubes and detection tubes, add reagents 1, 2, and 3 dropwise in sequence according to the instructions, mix well and then bathe in 60°C water for 15 minutes, then centrifuge at 3500 rpm for 10 minutes after cooling, take 200 μL of supernatant in a 96-well plate, and use a microplate reader Detect the absorbance value of each tube at a wavelength of 550nm, calculate the hydroxyproline content (ug/mg) in the lung tissue of each group of mice according to the following calculation formula, and perform statistical analysis; the calculation formula is as follows:

(5) Real-time fluorescent quantitative PCR reaction: Weigh the lung tissues of mice in each group, cut them into pieces on ice, add RZ lysate, and homogenize them. The concentration and purity of the total RNA in the sample were measured by a spectrophotometer; according to the TaKara reverse transcription operation manual, the extracted mRNA was reverse transcribed into cDNA, the concentration of the cDNA product was 0.5 μg/10 μL system, and the reverse transcription conditions were 37°C, 15min, 85°C, 5s; then perform real-time fluorescent quantitative PCR reaction to detect the expression of α-SMA (α-smooth muscle actin) and HMGCR (3-hydroxy 3-methylglutaryl-CoA reductase) in the lung tissue of mice in each group at the transcriptional level Differences, data processing using 2 ^{- △ △ Ct} relative quantitative analysis of the target gene;

The primer sequences are detailed in the table below:

Primers for real-time fluorescent quantitative PCR

(6) Immunohistochemistry: Paraffin sections of mouse lung tissue in each group were dewaxed with xylene, twice for 10 minutes each time, gradient alcohol dehydration: with 100% alcohol, twice for 5 minutes each time, with 95% alcohol, 85% alcohol Alcohol, 75% alcohol, 50% alcohol, once each, 5min; heat repair with sodium citrate (PH=6) antigen for 15min, then block with goat serum for 10min, incubate α-SMA/HMGCR primary antibody overnight, HRP Incubate with labeled goat anti-rabbit/anti-mouse secondary antibody for 30 minutes, develop color with DAB for 6 minutes, stain with hematoxylin for 20 seconds, rinse with running water for 2 minutes, dehydrate, make transparent, seal the slide (same as HE staining), take pictures with a 10x objective lens under an ordinary light microscope, and observe For the expression of α-SMA and HMGCR protein in each group, the positive area was counted by Image-ProPlus6.0, and the results were quantified by the ratio of the IOD value of the positive area to the area of the image;

(7) Statistics: SPSS20.0 software was used for statistical analysis, and the data of each group were expressed as mean ± standard deviation (x ± s), and the mean comparison among multiple groups was carried out by one-way analysis of variance, and the pairwise comparison between groups was carried out by LSD method, the test level is α = 0.05.

The feeding conditions of the adaptive feeding are 12 hours of light, 12 hours of darkness, temperature (23±1)° C., relative humidity of 50% to 65%, and independent drinking and eating.

_The concentration of the SiO suspension is 50mg/mL.

The concentration of CoQ ₁₀ is 0.2 mg/mL.

The HE staining steps are: (1) dewaxing: paraffin sections are dewaxed with xylene, twice for 10 min each; (2) gradient alcohol dehydration, using 100% alcohol, twice for 5 min each, using 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, each time for 5 minutes; (3) Distilled water washing for 3 minutes; (4) Hematoxylin staining for 6 minutes; (5) Distilled water washing for 10 seconds; (6) Hydrochloric acid ethanol differentiation for 5 seconds; (7) ) distilled water for 3 min; (8) eosin staining for 5 min; (9) distilled water for 5 s; (10) 85% alcohol once for 15 s, 95% alcohol once for 15 s, 100% alcohol twice for 3 min each; Twice with toluene for 3 minutes each time; (12) Seal the slide with neutral gum and examine with an optical microscope.

The Sirius Red staining steps are as follows: (1) dewaxing: paraffin sections are dewaxed with xylene, 2 times for 10 minutes each time; (steps are the same as HE staining) (2) Sirius Red staining solution is drip-stained for 40 minutes; (3) distilled water rinses After 2 minutes, the surface staining solution was removed, dehydrated and transparent (same as HE staining); (4) Mount the slide with neutral gum, and observe the fibrosis of the mouse lung tissue.

As shown in Figure 1, the alveolar structure of the lung tissue of mice in the control group was basically complete, the alveolar wall was not significantly thickened, and inflammatory cells did not appear to gather significantly; the lung tissue structure of the model group was significantly damaged, and a large number of inflammatory cells in the lung interstitium and alveolar cavity gathered , silicon nodules formed and larger in diameter; in the treatment group, there were accumulations of inflammatory cells, but the alveolar wall structure was acceptable, and silicon nodules appeared, but there were fewer nodules and smaller diameters.

As shown in Figure 2, the alveolar structure of the mice in the control group was normal, and there was no interstitial collagen deposition; the collagen content in the lung tissue of the mice in the model group was significantly increased, the collagen deposition in the alveolar interstitium was significantly thickened, and the alveolar structure was destroyed; Collagen deposition also appeared in the tissue, but compared with the model group, the fibrosis was alleviated, and the alveolar structure was acceptable.

In order to verify the differences in the content of hydroxyproline in the lung tissue of each group of mice, the present invention uses the alkaline hydrolysis method to detect, and the results of variance analysis between the three groups are F=5.280, P=0.030, and the results are shown in the following table: model group hydroxyproline The content of hydroxyproline in the treatment group was significantly higher than that in the control group (P=0.041), and the content of hydroxyproline in the treatment group was significantly lower than that in the model group (P=0.013).

Effect of CoQ10 on HYP content in mouse lung tissue ( n=8)

group HYP content (ug/mg) control group 0.544200±0.02775 model group 0.64575±0.028026 therapy group 0.547375±0.025605

The present invention adopts the qRT-PCR method to detect the mRNA expression difference of α-SMA in the lung tissue of each group of mice, and the result of variance analysis among the three groups is F=4.823, P=0.015. The results are shown in the table below: the mRNA expression level of α-SMA in the model group was significantly higher than that in the control group (P=0.048); the mRNA expression level of α-SMA in the treatment group was significantly lower than that in the model group (P=0.004).

Effects of CoQ10 on the expression of α-SMA mRNA and protein in lung tissue of mice ( n=8)

Immunohistochemical method was used to detect the expression of α-SMA protein in each group, and the brown color represented the positive area. Observed under the microscope: the expression level of α-SMA in the model group was significantly higher than that in the control group, while the expression level of α-SMA in the treatment group was significantly lower than that in the model group. After further statistics using IPP software, it was found that the expression of α-SMA in the lung tissue of the mice in the model group was significantly higher than that in the control group (P=0.008), while the expression of α-SMA in the lung tissue of the mice in the treatment group was higher than that in the normal saline group, no statistics However, the expression of α-SMA in the lung tissue of mice in the model group was significantly lower (P=0.005). The results are shown in Figure 3.

As shown in Figure 4, the expression of HMGCR protein in each group was detected by immunohistochemical method, and the brown color represents the positive area. The expression of HMGCR in the model group was significantly higher than that in the control group, and the expression of α-SMA in the treatment group was significantly lower than that in the model group and higher than that in the control group. group, using the IPP software to further count the positive areas of each group, and then perform variance analysis, there is a significant difference between the groups, F=9.575, P=0.002; The expression of HMGCR in mouse lung tissue was significantly lower than that of the model group (P=0.041), but the expression was significantly higher than that of the control group (P=0.022).

The present invention proposes that if silicosis mice are treated with CoQ ₁₀ , after 60 days, the HE staining results show that the alveolar structure of the model group is severely damaged, and inflammatory cells gather, while the treatment group has less inflammatory cell aggregation and alveolar structure than the model group. Can. Sirius red dye is a strongly acidic anionic dye, which can react with alkaline collagen to make collagen fibers appear red, and is widely used to detect the distribution of collagen in fibrotic tissues. The results of this study showed that the distribution of fibrotic collagen in the model group mice was significantly higher than that of In the control group, the collagen distribution level in the treatment group was lower than that in the model group but higher than that in the control group. Hydroxyproline is a non-essential amino acid unique to collagen, which can be used to evaluate the activation degree of fibroblasts. The present invention uses the alkaline hydrolysis method to test the hydroxyproline content in the lung tissue of mice in each group, and it was found that the model group was significantly higher than The control group and the treatment group were significantly lower than the control group and higher than the control group, which was consistent with the results of Sirius red staining. In order to further detect whether fibrous foci are formed during the fibrosis process, the marker α-SMA of myofibroblasts was detected by immunohistochemistry, and it was found that the expression of α-SMA in the model group was significantly higher than that in the control group, while the expression of α-SMA protein in the treatment group was significantly higher than that in the control group. The expression was significantly lower than that in the model group. The above results all indicated that CoQ ₁₀ had a certain anti-silicosis fibrosis effect.

CoQ ₁₀ is an important component of the hydrogen delivery complex on the inner mitochondrial membrane, which plays a vital role in the generation of cellular energy metabolism. HMGCR is the rate-limiting enzyme for the autonomous synthesis of CoQ ₁₀ in organisms. The study found that the abnormal expression of collagen I and fibronectin was detected in ectopic fat deposition (EFD) in the kidney, and they also found that the expression of HMGCoA (p-HMGCR) was also abnormal, which indirectly indicated that there was a relationship between HMGCR and collagen I; The chemical method found that the expression of HMGCR protein in the model group was significantly and stably higher than that of the control group, while the expression of HMGCR in the CoQ ₁₀ treatment group was lower than that of the model group but higher than that of the control group, which may be due to the damage of lung tissue cells caused by SiO ₂ inhalation into the lung tissue , Destroy the oxidative respiratory chain in tissue cells and reduce ATP synthesis. The body regulates the compensatory increase of HMGCR to promote the synthesis of endogenous CoQ ₁₀ and strengthen its own oxidative respiration level. However, when a certain amount of CoQ ₁₀ is given to the outside world, it may feedback reduce the expression of HMGCR, restore the damaged respiratory chain, and maintain the balance of energy metabolism in tissue cells , to further play the role of anti-fibrosis.

In summary, CoQ ₁₀ inhibits SiO ₂ -induced silicosis fibrosis, CoQ ₁₀ can down-regulate the expression of collagen, hydroxyproline and α-SMA, and its mechanism of action may be feedback regulation of endogenous HMGCR expression, affecting oxidative respiration chain. The present invention clarifies the influence of CoQ ₁₀ on HMGCR in silicosis fibrosis, and provides a new perspective for the study of the mechanism of subsequent silicosis treatment.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are included within the protection scope of the present invention.

Claims (6)

1.CoQ₁₀Application in anti-silicotic fibrosis, which comprises the following steps:

(1) reference group is set: choosing 6 week old male C57 of SPF grade mouse 24,20 ± 2g of weight, first adaptive feeding is after a week It is randomly divided into control group, model group, treatment group；Model group, treatment group are using intratracheal disposable instillation 0.1mL SiO₂Suspension Mouse Silicotic model is constructed, control group is the same as the isometric physiological saline of method instillation；0.1mL is given daily after the postoperative 48h for the treatment of group CoQ₁₀, stomach-filling, control group, model group are independently fed；

(2) Silicotic model is prepared: after the anesthesia of mouse isoflurane, exposure tracheae, respectively at control group instillation 0.1mL physiology salt Intratracheal instillation 0.1mL SiO distinguishes in water, model group and treatment group₂Suspension, then skin suture, observes mouse survival state； The postoperative 48h for the treatment of group, every mouse give 0.1mL CoQ daily₁₀Stomach-filling；After 60 days, infused with 0.3mL0.3% urethane abdominal cavity It penetrates anesthesia and puts to death mouse, take the middle lobe of right lung, then fixed with 4% paraformaldehyde with after normal saline flushing, remaining lung tissue is put into- 80 DEG C of refrigerators freeze；

(3) HE dyeing and sirius red stains: after fixing middle lobe of right lung 48h with 4% paraformaldehyde, being dehydrated, embed, slice, Slice thickness is 8 μm, carries out HE dyeing and sirius red stains after piece 6h is baked under the conditions of 56 DEG C；

(4) hydroxyproline determination: accurately weighing the mouse lung tissue that 50mg freezes with balance, shred on ice, and 1mL hydrolysis is added Liquid, water-bath under the conditions of 96 DEG C are fully hydrolyzed 20min and adjust each pipe PH after flowing water is cooling in 6.0-6.8, add distilled water extremely 10mL is mixed, and proper amount of active carbon is added, is centrifuged 10min under the conditions of 3500rpm, supernatant 1mL is taken to detect；Blank is set respectively One, two, No. three reagent is successively added dropwise in pipe, standard pipe and detection pipe, after mixing under the conditions of 60 DEG C water-bath 15min, it is cooling after It is centrifuged 10min under the conditions of 3500rpm, takes 200 μ L supernatants in 96 orifice plates, detects each pipe at wavelength 550nm using microplate reader and inhales Shading value calculates each group mouse lung tissue hydroxyproline content, and for statistical analysis；

(5) real-time fluorescence quantitative PCR reacts: each group mouse lung tissue is weighed, is shredded on ice, addition RZ lysate, and homogenized, The total mRNA of each group mouse lung tissue is extracted, the concentration and purity of micro-spectrophotometer measurement sample total serum IgE are used；By extraction MRNA reverse transcription is cDNA, and cDNA production concentration is 0.5 μ g/10 μ L system, and reverse transcription condition is 37 DEG C, 15min, 85 DEG C, 5s； Carry out real-time fluorescence quantitative PCR reaction again, detection each group mouse lung tissue α-SMA, HMGCR transcriptional level differential expression, Data processing uses 2^-△△CtRelative quantitative assay is carried out to target gene；

(6) immunohistochemistry: the paraffin section of each group mouse lung tissue is dewaxed with dimethylbenzene, 2 each 10min, gradient wine Essence dehydration: with 100% alcohol, 2 each 5min, with 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, each 1 time, 5min；15min is answered with sodium citrate (PH=6) antigen hot repair, then closes 10min with lowlenthal serum, by α-SMA/HMGCR primary antibody Be incubated overnight, HRP label goat antirabbit/anti-mouse secondary antibody be incubated for 30min, DAB develop the color 6min, haematoxylin dyeing 20s, flowing water punching 2min, dehydration, transparent, mounting are washed, the common lower 10 times of object lens of light microscopic microscope take figure, observe each group α-SMA and HMGCR albumen table Up to situation, positive region is counted with Image-Pro Plus 6.0, as a result with the ratio of positive region IOD value and picture area into Row is quantitative；

(7) it counts: being analysed using 20.0 software statistics credit of SPSS, each group of data is with mean ± standard deviationIt indicates, multiple groups Between mean compare and carried out using one-way analysis of variance, compare two-by-two between group using LSD method, inspection level is α=0.05.

2. CoQ according to claim 1₁₀Application in anti-silicotic fibrosis, it is characterised in that: the adaptive feeding Rearing conditions be 12h illumination, 12h is dark, temperature (23 ± 1) DEG C, relative humidity 50%~65%, autonomous drinking-water and feed.

3. CoQ according to claim 1₁₀Application in anti-silicotic fibrosis, it is characterised in that: the SiO₂Suspension Concentration is 50mg/mL.

4. CoQ according to claim 1₁₀Application in anti-silicotic fibrosis, it is characterised in that: the CoQ₁₀Concentration For 0.2mg/mL.

5. CoQ according to claim 1₁₀Application in anti-silicotic fibrosis, it is characterised in that: the HE staining procedure Are as follows: (1) dewax: paraffin section dewaxes through dimethylbenzene, 2 each 10min；(2) gradient alcohol dehydration, using 100% alcohol, 2 Secondary each 5min, using 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, each 1 each 5min；(3) distillation washing 3min；(4) haematoxylin dyeing 6min；(5) distilled water flushing 10s；(6) acidic alcohol breaks up 5s；(7) distilled water flushing 3min； (8) eosin stains 5min；(9) distilled water flushing 5s；(10) 85% 15s of alcohol 1 time, 95% alcohol, 1 15s, 100% alcohol 2 Secondary each 3min；(11) 2 each 3min of dimethylbenzene；(12) neutral gum mounting, Microscopy.

6. CoQ according to claim 1₁₀Application in anti-silicotic fibrosis, it is characterised in that: the Picro-Sirius red dye Color step position: (1) dewax: paraffin section dewaxes through dimethylbenzene, 2 each 10min；(2) sirius red stains drop contaminates 40min；(3) distilled water flushing 2min, removes surface dye liquor, and conventional dehydration is transparent；(4) neutral gum mounting observes mouse lung Tissue fibrosis changes.