CN114344387B

CN114344387B - Traditional Chinese medicine composition for treating pulmonary fibrosis diseases and application thereof

Info

Publication number: CN114344387B
Application number: CN202210085417.7A
Authority: CN
Inventors: 杨光辉; 潘新; 姚静慧; 胥晓芳; 何奕坤; 吴辉辉; 沈佳莹; 王运超
Original assignee: Shuguang Hospital Affiliated to Shanghai University of TCM
Current assignee: Shuguang Hospital Affiliated to Shanghai University of TCM
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-11-29
Anticipated expiration: 2042-01-25
Also published as: CN114344387A

Abstract

The invention relates to a traditional Chinese medicine composition for treating pulmonary fibrosis diseases, which is prepared from the following raw material medicines in parts by weight: 28-31 parts of radix rehmanniae, 29-32 parts of centella asiatica, 12 parts of curculigo orchioides, 12 parts of epimedium herb, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice. The invention also provides application of the traditional Chinese medicine composition. The invention takes the key pathogenesis of lung impediment, namely heat toxin stasis and obstruction of lung collaterals as the principle, has compact medication and precise and appropriate compatibility of medicines, strengthens body resistance to eliminate pathogenic factors, strengthens the foundation and clears the source. The invention can delay the decline of the lung function of a patient when being applied to clinic. In terms of FVC drop rate: the total effective rate of the Chinese and western medicine combination group is 51.52 percent, which is higher than that of a control group of single-use western medicine (pirfenidone) by 36.36 percent (P is less than 0.05); animal experiments prove that the invention can reduce the lung coefficient of a bleomycin filming rat, the fibrosis condition of a lung pathological section and the levels of KL-6, IL-21 and TGF-beta 1 of the rat. The invention proves that the traditional Chinese medicine composition has a certain improvement effect on pulmonary fibrosis.

Description

Traditional Chinese medicine composition for treating pulmonary fibrosis diseases and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating pulmonary fibrosis diseases and application thereof.

Background

Pulmonary Fibrosis (PF) is a heterogeneous group of diseases caused by different causes, mainly manifested by progressive shortness of breath, restricted ventilation dysfunction, diffuse hypofunction and hypoxemia, which finally results in respiratory failure and even death of patients, and the prognosis is poor. Clinical manifestations of PF, imaging manifestations, lung function and pathology due to different etiologies share many similarities and overlaps. PF may occur in most Connective Tissue Diseases (CTD), such as Rheumatoid Arthritis (RA), with varying rates and severity. Pulmonary fibrosis often progresses chronically and progressively, finally results in lung function loss, has high mortality, causes huge expenditure of social medical resources, and brings huge burden to patients and families. Therefore, the medicine for resisting pulmonary fibrosis is actively sought, and has profound social significance.

At present, the western medicine mainly selects the drugs of pirfenidone, nintedanib and the like for treating the pulmonary fibrosis, although the progress of the pulmonary fibrosis is delayed to a certain extent, the effect is not satisfactory, and the drugs are easy to generate the toxicity of liver and kidney after being taken for a long time, thereby seriously restricting the clinical application of the drugs. Its high price is prohibitive for many patients.

According to the traditional Chinese medicine, pulmonary fibrosis belongs to the category of lung impediment, and the lung impediment is abundantly placed in the internal channel: … … is used for treating lung diseases, named lung impediment, cough and qi. "; su Wen & Wu zang Sheng: ' Chuanfloating, deficient in the upper part and excessive in the lower part … … Chuanxu, named as Lung bi-arthralgia. "; the plain question-Bi Lun has a main expression: the "lung impediment" with restlessness, dyspnea and vomiting emphasizes the characteristics of the lung impediment "dyspnea"; the doctor of the past generations perfected and supplemented the concept of "lung bi-arthralgia", but basically it is not described in "Nei Jing", which defines "lung bi-arthralgia" as one of the five zang organs bi-arthralgia. The pathogenesis of the lung impediment is complex, and the traditional Chinese medicine considers that people with intense fire and heat have toxic materials, people with severe and persistent disease conditions and unclear etiology have toxic materials, people with complex pulmonary fibrosis have complicated disease conditions and complicated disease conditions, and the traditional Chinese medicine has multiple causes, and the traditional Chinese medicine is always related to heat and toxicity. The lung collaterals are damaged by toxicity, healthy qi is consumed and damaged, blood circulation is not smooth, blood stasis is generated, and cough and asthma are caused when the lung is attacked by stagnation. In a word, the pathogenesis of lung impediment is complex, and the lung impediment always refers to stasis and toxic impediment blocking lung collaterals, the disease location is deep, the disease condition is serious, and the treatment mainly aims at clearing lung toxicity, removing blood stasis and dredging collaterals.

Chinese patent document CN103933360A discloses a traditional Chinese medicine composition for treating pulmonary fibrosis, which is characterized by being prepared from the following raw material medicines by weight: 3-9 g of scutellaria baicalensis, 3-5 g of paris polyphylla, 6-8 g of mulberry leaves, 6-9 g of edible tulip, 3-5 g of plum blossom, 4-6 g of radix glehniae, 1.5-3 g of sweetgum, 6-9 g of semen lepidii, 10-15 g of dahurian rhododendron leaf, 6-9 g of andrographis paniculata, 3-5 g of herba aristolochiae, 6-8 g of emblic leafflower fruit, 3-5 g of lumbricus, 3-5 g of hairyvein agrimonia herb and bud and 6-9 g of fructus terminaliae.

However, no reports on the traditional Chinese medicine compositions for treating pulmonary fibrosis diseases such as radix rehmanniae, centella asiatica, curculigo orchioides, epimedium herb and the like are found at present.

Disclosure of Invention

The first objective of the present invention is to provide a Chinese medicinal composition for treating pulmonary fibrosis diseases, which overcomes the disadvantages of the prior art.

The second purpose of the invention is the application of the traditional Chinese medicine composition.

In order to achieve the first purpose, the invention adopts the technical scheme that: a traditional Chinese medicine composition for treating pulmonary fibrosis diseases is prepared from the following raw material medicines in parts by weight: 28-31 parts of radix rehmanniae, 29-32 parts of centella asiatica, 12 parts of curculigo orchioides, 12 parts of epimedium herb, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

As a preferred example, the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 30 parts of radix rehmanniae, 30 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

In order to achieve the second object, the invention adopts the technical scheme that: the application of the traditional Chinese medicine composition in preparing the medicine for treating pulmonary fibrosis diseases.

As a preferred example, rheumatoid arthritis may develop secondary pulmonary fibrosis.

The invention is clinically proved to have curative effect on delaying the progress of the rheumatoid arthritis related pulmonary fibrosis, and after the traditional western medicine is taken for 1"7 months, the HRCT of the chest of the same patient is compared on the same layer, so that the glass-like shadows are reduced compared with the cases before the medicine is taken, and the fibrosis is reduced. The invention sets up a bleomycin-induced pulmonary fibrosis rat model, and finds that the lung coefficient, the contents of KL-6, IL-6 and IL-21 in serum and the contents of KL-6, TGF-beta 1 and IL-6 in lung tissues can be effectively reduced in example 1. And the high-dose group and the low-dose group have better curative effect and no serious adverse reaction.

The invention aims to provide a clinical application recipe for treating pulmonary fibrosis. Pulmonary fibrosis belongs to the category of lung bi, and the Qing Gaoshi mountain of the treatment of symptoms of high-quality golden lack and blood bi consumptive disease says that the symptoms of bi and inferior qi are also known as bi, because the blood is deposited and the symptoms of qi weakness are known as bi. According to the subject group of the invention, years of clinical experience shows that the pathogenesis of lung obstruction is complex, and the lung obstruction generally belongs to phlegm, stasis and toxin obstruction and obstruction of lung collaterals. The nature of the disease is deficient of the principal, marked by excess, mixed with deficiency and excess. The disease is located in the lung and closely related to the spleen and kidney. In the interior Jing, the interior meridians, all of them are fire, and those with toxic pathogen are marked by violent qi in five groups, and the fire heat remains and remains latent to deep and accumulates into toxicity, i.e., the pathogenic factors are extremely toxic. Obstruction of meridians and collaterals, blood stagnation in vessels, and blood stasis. Heat-stasis can cause the stagnation of heat, which leads to toxic pathogen and finally to the accumulation of blood stasis. Wangqingren cloud: "Qi circulation without stagnation, blood circulation without stasis, qi circulation without blood stasis, and the patient's trouble. Therefore, the doctor of China Yan Dexin who says "qi is the length of a hundred diseases and blood is the fetus of a hundred diseases" advocate advocates "the theory that a long-term disease will have stasis and a strange disease will have stasis" advocates "the balance method, meaning" regulating qi and blood, regulating yin and yang "and by regulating qi and blood balance, achieves the purpose of treating pulmonary paralysis.

The invention has the advantages that: based on clinical treatment experience, the theory of viscera and qi and blood is combined, and a national doctor of great expertise Yan Lao 'Heng Fang' is used for treating the lung impediment, so that the principle of regulating qi and blood is taken as the basis, the lung and kidney are emphasized to be tonified, the principal and secondary aspects are considered, and the effects of clearing lung, removing blood stasis, dredging collaterals, eliminating phlegm and detoxifying are achieved all the time.

In the formula, dried rehmannia root, radix rehmanniae is sweet and cold in nature and enters kidney, nourishes yin and strengthens water, clears heat and cools blood, is bitter, pungent and cold in centella, can clear lung heat and decompose arthralgia-syndrome toxin, can activate blood and dissipate blood stasis, and is the monarch drug together with dried rehmannia root. The ministerial drugs adopt the curculigo orchioides and the epimedium herb to warm and tonify the kidney yang, intend to generate the golden water, dispel the wind-damp to remove the arthralgia-syndrome toxin, and help the rehmannia root to nourish water and consolidate the golden; peach kernel promotes blood circulation and removes blood stasis, relieves cough and asthma, and radix salviae miltiorrhizae promotes blood circulation and removes blood stasis, clears heat and cools blood, cooperates with centella to clear and remove stagnant heat; radix Dipsaci is used in combination with radix Scutellariae for promoting blood circulation, tonifying liver and kidney, preventing the excess of warm-natured medicines; the liquorice has the effects of clearing heat and removing toxicity, eliminating phlegm and stopping cough when used for harmonizing the raw materials, and has the peculiar effects of clearing lung and dissolving stasis when used together with the raw materials, the medicines are soft but not greasy, clear but not cold, activating blood without damaging vital qi, and treating both pathogenic factors and vital qi. The whole formula has compact medicine, precise and appropriate compatibility, strengthens body resistance to eliminate pathogenic factors, fixes foundation and clears source. The invention shows that the model is successful by observing the general condition of various groups of rats with pulmonary fibrosis, the pulmonary coefficient of the rats and the content change of KL-6, IL-21 and TGF-beta 1, and after the model is administrated, the pulmonary coefficient and the pulmonary pathology of the rats are improved to different degrees, and the content of KL-6, IL-21, TGF-beta 1 and the like is reduced to different degrees. The invention proves that the traditional Chinese medicine composition has a certain improvement effect on pulmonary fibrosis.

Drawings

FIG. 1 shows the same HRCT slice comparison of the same patient's chest after taking "example 1".

Figure 2 groups of lung tissue gross morphology a: sham group B: a module C is built: traditional Chinese medicine high dose group D: low dosage group of Chinese medicinal herbs

FIG. 3 Effect of groups of rat Lung tissue TGF-. Beta.1

FIG. 4 pathological conditions of lung tissue (HE staining, X200) in groups of rats and infiltration of alveolar septal inflammatory cells into ≠ blood vessels

FIG. 5 shows the expression of KL-6, TGF-. Beta.1, and IL-6 proteins in rat lung tissues of each group. Note: p < 0.05 compared to model group.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1

Weighing the following raw material medicines in parts by weight: 30 parts of radix rehmanniae, 30 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

Example 2

Weighing the following raw material medicines in parts by weight: 28 parts of radix rehmanniae, 31 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

Example 3

Weighing the following raw material medicines in parts by weight: 29 parts of radix rehmanniae, 32 parts of centella asiatica, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

Example 4

Weighing the following raw material medicines in parts by weight: 30 parts of radix rehmanniae, 28 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

Example 5

Weighing the following raw material medicines in parts by weight: 31 parts of radix rehmanniae, 30 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

Example 6 (control 1)

Weighing the following raw material medicines in parts by weight: 33 parts of radix rehmanniae, 30 parts of centella, 12 parts of rhizoma curculiginis, 14 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

Example 7 (control group 2)

Weighing the following raw material medicines in parts by weight: 30 parts of radix rehmanniae, 35 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 18 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 16 parts of dipsacus root and 6 parts of raw liquorice.

EXAMPLE 8 clinical efficacy test

The invention can be used for treating patients with rheumatoid arthritis-related pulmonary fibrosis for more than 10 years, and the clinical curative effect is definite.

The observed cases were: some Zhao, female, age 60, 12 months in 2020, was diagnosed with rheumatoid arthritis-associated interstitial lung disease. Example 1 (example 1) was taken on a regular treatment basis from 3 months 2021. By 10 months in 2021, after taking example 1"7, the same patient's chest HRCT had less glass-like shadows on the same layer than before administration, and fibrosis was significantly reduced.

Clinical efficacy of "example 1" was confirmed by clinical randomized controlled trials:

the study was approved by the ethical committee of clinical trials and completed registration in the registration center for clinical trials in china.

1 data and method

1.1 case selection

1.1.1 diagnostic criteria (1) refer to The diagnostic criteria for RA formulated by The American College of Rheumatology (ACR) in 2009, the European Association for Rheumatism (EULAR) (2) The diagnostic criteria for RA-ILD The American thoracic Association/European respiration Association (American Thoracic Society, ATS/European Respiratory Society, ERS). The published clinical diagnostic criteria for idiopathic pulmonary interstitial disease are equally applicable to The diagnosis of connective tissue disease and pulmonary interstitial disease, i.e. on The basis of The established RA, the clinical diagnostic criteria for idiopathic pulmonary interstitial disease proposed in 2000 ATS/ERS are also met, and can be diagnosed as RA-ILD. (3) The Chinese medicine diagnosis and syndrome differentiation standard is combined with the clinical diagnosis and treatment experience of the traditional Chinese medicine, and refer to the Chinese medicine industry standard of the people's republic of China-the Chinese medicine disease diagnosis and treatment efficacy standard (ZY/T0OI.1-94), the disease belongs to Wangbi (internal housing in lung), and the syndrome belongs to lung heat and blood stasis, and the symptoms are as follows: arthralgia, shortness of breath, weakness, dry cough with no or little sputum, dry mouth and throat, dark tongue with little fluid and little coating, or thin and white coating, and thready and rapid pulse.

1.1.2 inclusion criteria (1) meets diagnostic criteria and Chinese medicine dialectical criteria (2) 18 to 80 years of age (3) informed consent (4) RA stable, defined as: no new or replacement of RA treatment regimen was started within 6 weeks prior to enrollment and was judged by the rheumatology specialist as RA reaching standard (5) progression of pulmonary fibrosis, defined as: on the premise of excluding changes attributable to complications (e.g., infection, heart failure), at least one of the following criteria was met 24 months prior to enrollment: the predicted value of the FVC is reduced clinically, namely the relative reduction is more than or equal to 10 percent; II, a critical decline appears in the predicted value of the FVC, namely, the relative decline is more than or equal to 5 and less than 10 percent, and respiratory tract symptom exacerbation is combined; III, critical decline of the FVC predicted value occurs, namely, the relative decline is more than or equal to 5 and less than 10 percent, and the change range of the fibrosis is enlarged by combining with the chest imaging examination; worsening respiratory symptoms and increasing range of changes in breast image fibrosis (6) patients who have not been recently treated with anti-fibrosis or who have been washed out. (7) Combining the medicines: before the administration, the patient should take methotrexate and leflunomide for more than 3 months or stop taking the medicine for more than 1 month; the hormone or biological preparation can be administered after stopping administration for more than 3 months

1.1.3 exclusion criteria (1) severity staging criteria for IPF disease, as established by the Japanese society of respiratory disease, in stage IV; or pulmonary interstitial fibrosis caused by other reasons of a patient (2) who cannot complete the pulmonary function test due to illness before treatment and idiopathic pulmonary interstitial fibrosis; or those with lung infection, lung cancer, tuberculosis (3) significant pulmonary hypertension as defined by any of the following criteria: clinical or echocardiographic evidence of previously significant right heart failure; ii, the medical history includes that the right heart catheter shows that the heart index is less than or equal to 2L/min/m ² (ii) a Eprostenol/treprostinil parenteral treatment (4) pregnant or lactating women (5) allergic to the drug used (6) patients with serious cardiovascular, hepatic, renal, hematopoietic disorders (7) patients with psychiatric disorders (8) who are recently attending other clinical trials

1.1.4 falling-off standard (1), 2, patients who quit from the hospital without finishing the course of treatment, 3, the data of which are not complete, influence the curative effect and the safety judgment, and the patients (4) are not suitable to be continuously treated by the testee (5), and the patients with tuberculosis infection, septicemia, demyelinating lesion and viral hepatitis appear.

1.1.5 rejection criteria (1) No use of test drug (2) against the prescription of concomitant medication, and No data after administration of drug (3) affecting the judgment of efficacy and safety

1.2 general data all cases were from the rheumatological and immunologic department outpatient clinic of the eosin hospital affiliated with university of medicine in Shanghai, 6 months to 2021 month in 2020, with a total income of 66, of which 25 were male and 41 were female; minimum age 35 years, maximum age 78 years, mean (65.64 ± 8.80) years; mean course (123.19 ± 117.802) months. According to a clinical trial randomization scheme, a random grouping envelope is generated by adopting an SPSS25.0 software random number table, the random grouping envelope is sequentially put into a subject according to a certain sequence, and the subject randomly enters a traditional Chinese medicine group and a control group according to numbers in the envelope. Because the incidence rate of rheumatoid arthritis is higher for women than for men, the number of women in patients who are grouped at this time is larger. Through statistics, the gender, age and course of disease of the patients in the two groups have no statistical difference (P is more than 0.05), and the patients have comparability, which is shown in table 1.

TABLE 1 general information

1.3 intervention method according to '2018 diagnosis of related interstitial lung disease of connective tissue disease and consensus of therapist', patients with RA remission and ILD progression can maintain immunosuppressive therapy and increase anti-fibrosis therapy, and anti-fibrosis drugs mainly comprise pirfenidone, nintedanib and the like. (1) Control group: RA remission treatment regimen + pirfenidone (200 mg-400mg tidpo, with increasing doses from 200mg, depending on patient tolerance); (2) treatment groups: addition of "example 1" to the control group "

1.4 Observation project and method

1.4.1 efficacy index (1) main efficacy observation index pulmonary function reference international universal clinical endpoint index, the main efficacy index of this study is forced vital capacity change rate (Δ FVC), patient FVC is measured before and after treatment, and FVC change rate before and after treatment is calculated: Δ FVC = (FVC after treatment-FVC before treatment)/FVC before treatment × 100% (2) lung HRCT semi-quantitative integral reference "clinical CT diagnostics", the images were diagnosed by radiologist and clinician, the scoring subjects were defined as ground glass-like shadow or solid shadow, reticular shadow (including leaflet space thickening, leaflet interstitial thickening, bronchial vascular bundle thickening, pleural offline), alveolar lung, bronchiectasis, and semi-quantitative scoring was performed according to the area ratio of the scoring subjects on the scanning planes (6 planes in total of 1cm on the top of the lung tip, lung portal, aortic arch, carina, right inferior pulmonary vein and right diaphragm), and the total score was 24: if no obvious sign on the scanning layer is displayed as 0 point; the aspect proportion of the signs is 1 point when the aspect proportion is less than 25 percent; 25-50% of the total weight is 2 minutes; 50% -75% is 3 minutes; > 75% for 4 min. The consensus given by two doctors after discussion is the final result of reading the lung HRCT semi-quantitative integral measured before and after treatment. (3) According to the Chinese medicine syndrome integration referred to the Chinese medicine new drug clinical research guideline (2002), and the Chinese medicine syndrome integration component table with good reliability and validity, the symptoms of cough, expectoration, short breath, asthma, chest distress, dry throat, cyanosis and the like of a patient are evaluated and counted for 0, 1, 2 and 3 according to no, light, medium and heavy 4 grades respectively, and the tongue and pulse conditions are only recorded and not counted. The Chinese medicine syndrome integrals are measured before and after treatment.

1.4.2 Total curative effect assessment Standard clinical Total curative effect assessment Total curative effect is assessed according to the after-treatment FVC change rate according to the consensus and guidelines of the relevant experts, showing effects: FVC change rate is > -5%; the method has the following advantages: the FVC change rate is less than or equal to-5% and is greater than-10%; and (4) invalidation: the FVC change rate is less than or equal to-10%, or the lung function test can not be completed again due to the disease condition. The criteria for judging the therapeutic effect of syndromes are calculated according to the nimodipine method with reference to the guidelines of clinical research on new Chinese drugs (2002). The effect is shown: the reduction of syndrome integral is more than or equal to 70 percent; can normally participate in work and labor; the method has the following advantages: the reduction of syndrome integral is more than or equal to 30 percent; and (4) invalidation: the syndrome integral is reduced by less than 30%. Total effective rate = (number of effective cases + number of effective cases)/total number of cases × 100%.

1.4.3 adverse reaction assessment patients were monitored during the trial for alanine Aminotransferase (ALT), blood creatinine (SCR) to assess the presence or absence of impairment of liver and kidney function; the patients after taking the medicine are observed whether the new digestive tract symptoms and anaphylactic reaction appear, and the symptoms are different from the symptoms of the disease, and the observation and the recording are needed at any time.

1.5 statistical methods SPSS 26.0 statistical software was used for analysis. If the measured data conforms to the normal distribution, the measured data is described by mean plus or minus standard deviation (x plus or minus s), and if the measured data does not conform to the normal distribution, the measured data is described by median (quartile distance) [ P50 (P25, P75) ]. Difference between the measured data groups is compared before and after the test, if the data accords with normal distribution and the variance is uniform, two independent samples are adopted for t test, and if the variance is not uniform, two independent samples are adopted for t' test; if the data does not conform to normal distribution, a Mann-Whitney U rank sum test is used. Repeated measurements (at equal time intervals) were analyzed using variance analysis of the repeated measurements. Counting data are described by using the number (percentage) of [ n (%) ], the difference between groups is compared by using a chi-square test or fisher's test, and the test level is corrected by using a Bonferroni method. The difference is statistically significant when P is less than 0.05.

2 results

2.1 the total curative effect and the traditional Chinese medicine syndrome curative effect are compared and compared with the total curative effect of two groups in delta FVC, the total effective rate of the traditional Chinese medicine is 51.52 percent, the total effective rate of the control group is 36.36 percent, and the difference has statistical significance (X2 =8.618, P =0.004 < 0.05), which is shown in Table 2. After treatment, the curative effects of the two groups of traditional Chinese medicine symptoms are compared, the effective rate of the traditional Chinese medicine group is 33.33%, the effective rate of the control group is 69.70%, and the difference has statistical significance (X2 =8.738, P =0.013 < 0.05), which is shown in Table 3.

TABLE 2 Overall efficacy comparison (example)

* P < 0.05 in comparison with the control group

TABLE 3 comparison of the curative effects of the syndromes in traditional Chinese medicine

* P < 0.05 in comparison with the control group

2.2FVC changes prior to treatment, the FVC differences between the two groups of patients were not statistically significant (P =0.448 > 0.05). After treatment, the comparison in the group shows that the FVC in the control group and the traditional Chinese medicine is reduced compared with that before treatment, and the difference has statistical significance (P = < 0.001 and P = < 0.025 < 0.05); compared with the control group, the traditional Chinese medicine FVC is higher, and the difference has statistical significance (P =0.011 < 0.05). The results show that the degree of FVC reduction of the patients in the traditional Chinese medicine group is reduced compared with the control group, and the results are shown in Table 4.

2.3 pulmonary HRCT semi-quantitative score before treatment for changes in pulmonary HRCT semi-quantitative score the difference in pulmonary HRCT semi-quantitative score was not statistically significant in both groups (P =0.977 > 0.05). After treatment, in-group comparison, the HRCT score for the control group and the traditional Chinese medicine lung had no statistical significance with the difference before treatment (P =0.156 >0.05, P =0.153 > 0.05), indicating that the HRCT semi-quantitative score for both groups of lungs did not change significantly within a half-year treatment period. See table 4.

TABLE 4 semi-quantitative integral comparison of FVC and pulmonary HRCT in two groups before and after treatment: (

n＝33)

* P is less than 0.05 compared with the group before treatment; p is less than 0.05 after the treatment of the tangle-solidup root and the contrast group; # P >0.05 compared to pretreatment of this group;

2.4 before the change of the traditional Chinese medicine syndrome integrals is treated, the two groups of traditional Chinese medicine syndrome integrals are compared, and the difference has no statistical significance (P =0.26 > 0.05). After treatment, the traditional Chinese medicine syndrome integration of the control group and the traditional Chinese medicine is reduced compared with that before treatment (P is less than 0.001; compared with the group, the traditional Chinese medicine group has lower Chinese medicine syndrome-waiting score than the control group, and the difference has statistical significance (P =0.04 < 0.05). The two groups can improve the traditional Chinese medicine syndrome of the patient, and the traditional Chinese medicine group has better effect of improving the traditional Chinese medicine syndrome than the control group.

TABLE 5 comparison of the Chinese medicine syndrome/score change conditions

Minute)

* P is less than 0.001 compared with the group before treatment; p is less than 0.05 after the treatment of the tangle-solidup root and the contrast group;

2.5 adverse reaction evaluation of patients with no liver injury in both groups before treatment (ALT > ULN, upper limit of normal), ALT differences were not statistically significant (P > 0.05). After treatment, 3 patients with liver injury in the control group and 1 patient with liver injury in the traditional Chinese medicine group are treated. During the treatment process, patients in the traditional Chinese medicine group have no obvious discomfort, and 3 cases in the control group have gastrointestinal symptoms such as slight abdominal distension and diarrhea. No renal function injury occurs before and after treatment of the two groups of patients. The total adverse reaction incidence rate of the traditional Chinese medicine is lower than that of a control group (P is less than 0.05).

Example 9 animal experiments

The invention can relieve the progress of pulmonary fibrosis of rats caused by bleomycin molding

1 materials and methods

1.1 materials

1.1.1 animals male SD rats of SPF grade 40, with body mass (200. + -. 20 g) purchased from Kyoho Calvens laboratory animals, inc. Animal production license number: SCXK (threo) 2016-0010. The animal experiment center of the transformation medicine research institute of Shanghai university is bred, and the experimental animals use license numbers: SYXK (Shanghai) 2014-0016. The feeding conditions comprise room temperature of 20-25 ℃, humidity of 40-70%, standard feed, free diet of animals and drinking water. SD rats were housed in a pathogen-free SPF grade animal room for 1 week.

1.1.2 drugs and reagents the present invention, example 1, extracted by water extraction and concentrated to the maximum concentration (based on the injection with a gavage needle) capable of gavage administration, corresponds to 1.42g of crude drug per ml (based on the equivalent dose ratio table of human and animal converted from body surface area, the administration dose of rat converted from clinical dose, example 1 group administration dose is 14.19g/kg body mass).

Methylprednisolone powder (methylprednisolone sodium succinate for injection, 40 mg/count), framexi-Puqiang Kalama Zu Fenan (batch number: 160 JTB); bleomycin hydrochloride (Bleomycin, 15 mg/liter) for injection, nippon Kagaku K.K. (batch No. J162310); ketamine hydrochloride injection (50 mg/mL), new gang pharmaceutical factory (batch number: 160408) of Shanghai Chinese and Western medicine industry, inc.; diazepam injection (5 mg/mL), shanghai Xue Donghai general pharmaceutical Co., ltd (batch No: 160501); sialorrhea melting sugar chain antigen (krebs von Lungen-6, KL-6), interleukin 6 (interleukin-6, IL-6), interleukin 21 (interleukin-21, IL-21), transforming growth factor-beta 1 (transforming growth factor-beta 1, TGF-beta 1) detection kit (Nanjing institute of bioengineering research)

1.1.3 instrumental microtomes, leica, germany (model: RM 2145); superclean bench, singapore ESCO corporation (model: SVE-6A 1); medical high speed cryogenic centrifuges (Du Pont T21, duPont, USA); a decoloring shaking table and a new wave wireless power plant (type: TY-B) in Shanghai city are used; bio-Rad electrophoresis apparatus, shanghai medical Analyzer apparatus factory (model: DY-1); a hybridization oven, shanghai chromatography Instrument Co., ltd. (model: robbin Scientific 1000); gel imaging System (model: gel-Doc EQ), bioRad Inc. of USA; fluorescent inverted microscope, OLMYPUS, japan (model: CKX41/U-RFLT 50).

1.2 grouping and modeling SD rats were randomly divided into 4 groups, i.e., a sham operation group, a model group, a high dose group (high dose group) in example 1, and a low dose group (low dose group) in example 1, 10 per group, according to a random number table method.

The bleomycin intratracheal injection method is adopted to replicate a rat pulmonary fibrosis model: diazepam (20 mg/kg body mass) is injected into the abdominal cavity, and chlorthalidone (20 mg/kg body mass) is injected into the abdominal cavity for anesthesia. After anaesthesia, the rat is fixed in the supine position, after disinfection, the skin is cut on the neck along the median line, the soft tissue is separated in a blunt way layer by layer, and the trachea is exposed. Inserting a 1mL syringe with plastic catheter, from which bleomycin 0.9% of NaCl solution (8 mg/kg body mass) has been extracted, into trachea via tracheal cartilage, slowly injecting 0.3mL of medicinal liquid, immediately rotating the rat vertically, and assisting the rat in vertical and rotational movements so that the drug is uniformly distributed in the lung. The incision is closed and the local skin is treated to resist infection. After the animals are naturally awake, the animals are raised in a cage, and the phenomena of lackluster hair, piloerection, slow response, body temperature reduction, slow or reduced physical quality increase, crouching and the like of the rats appear on the 7 th day are identified to establish the model to be successfully made. The animal model making method of the sham operation group is the same as the above, but the physiological saline is only used for replacing the bleomycin to carry out the intratracheal injection.

1.3 intervention administration method rat dose is converted by clinical dose according to the equivalent dose ratio table of human and animal converted by body surface area. The group of example 1 was administered at a dose of 14.19g/kg body mass and formulated to have a concentration of 1.42g/mL. Example 1 groups were administered daily at respective concentrations example 1 with a high dose of 10mL/kg body mass, a low dose of 5mL/kg body mass, and sham and model groups with a 0.9% NaCl solution of 10mL/kg body mass. The preparation is administered 1 time daily for 4 weeks. Intervention was started on day 1 after molding of each group until the end of the experiment.

1.4 detection index and method

1.4.1 general conditions and lung tissue general observations after 4 weeks of drug intervention, rats were weighed and observed for hair, mental status and drinking water. 10% chloral hydrate (3 mL/kg) is injected into abdominal cavity for anesthesia, the rat is fixed in the supine position, the abdominal cavity is opened, the incision is prolonged, the thoracic cavity is opened, the trachea is cut off at the position close to the thyroid cartilage, and the heart and the lung are completely taken out. Firstly, the heart and the thymus are removed and the whole lung tissue is taken out, and the general shape, the elasticity, the blood stasis and the like of the lung tissue are observed.

1.4.2 Lung factor the lungs removed were rinsed clean with 0.9% NaCl solution, blotted dry on filter paper, and weighed on a microbalance. Lung coefficient = wet lung weight (g)/mass of rat at sacrifice (g)

1.4.3 serum KL-6, IL-6 and IL-21 contents were collected from the abdominal aorta of rats, injected into a centrifuge tube, centrifuged at 4 ℃ for 20min (3000 r/min), and the serum was aspirated and sealed in a pyrogen-free test tube. The contents of KL-6, IL-6 and IL-21 in the rat serum are detected by ELISA method, and the operation steps are according to the kit instructions.

1.4.4 Lung tissue TGF-. Beta.1 content about 3mg of the lung tissue sample was sheared on a microbalance, 0.9% NaCl solution 1mL was added to a clean biological tissue mortar, the lung tissue sample was placed, liquid nitrogen was repeatedly added, the soil was rapidly ground in liquid nitrogen until the tissue was completely crushed, the tissue homogenate in the mortar was transferred to a clean test tube by a pipette, the mortar was rinsed 2 times with 0.9% NaCl solution 1mL, the rinse was transferred to a clean test tube, centrifugation was carried out at a low temperature of 2000r/min for 10min in total, the supernatant was aspirated by a pipette into a clean-tipped tube, 0.5g of the sample was put into a glass tube, 0.5mL of the tissue extract was added, the glass tube was placed in a 110 ℃ oven, hydrolyzed for 6 to 12h, 00r/min,25 ℃, centrifugation was carried out for 20min, the amount of 1605 mL was determined by constant volume using the extract, the supernatant was taken, and the lung tissue-. Beta.1 content was measured by ELISA.

1.4.5 HE staining of Lung tissue A part of the right lobe of the lung was excised, fixed with formaldehyde, paraffin embedded, and sliced at 4 μm for conventional HE staining: paraffin section xylene dewaxing 2 times, 12 min/time, adding ethanol gradient (100%, 95%, 80%, 70%) into water, hematoxylin staining for 6min, blue-staining with tap water for 10min, performing hydrochloric acid ethanol differentiation according to nuclear staining depth, eosin staining for 3min, dehydrating with ethanol gradient, xylene transparence, and sealing with neutral gum. And (5) observing the inflammation of lung tissues and the proliferation degree of collagen fibers by microscopic examination.

1.4.6 pulmonary tissue KL-6, IL-6 and IL-12 protein expression the expression of the pulmonary tissue KL-6, IL-6 and IL-12 proteins was detected by Western Blot method. Buckling the prepared SDS-PAGE gel plate on an electrophoresis tank, adding 1 × Running Buffer, pulling out a comb, adding a protein Marker and a sample, 80V,30min;120V,70min electrophoresis. The film transfer tank was placed in an ice-water mixture at 300mA for film transfer at 1h. After blocking with 5% skim milk, an antibody dilution containing the antibody of interest was added and incubated overnight at 4 ℃. Washing the membrane for 5min on a high-low shaking table for 3 times by using TBST for the next day; adding an antibody diluent containing a second antibody, and incubating at room temperature for 1-2 h; and finally, preparing A, B liquid of the ECL color developing liquid according to the ratio of 1: 1, and coating the ECL color developing liquid in a tin foil paper to prevent light. The membrane was placed on a test plate, the residual TBST was carefully aspirated off, about 200. Mu.L of developing solution was added, and the membrane was wiped off after spreading uniformly. The secondary antibody was incubated for 2h at room temperature every other day, followed by luminescence photography with a gel imager and analysis of band gray values with Image J.

1.5 statistical methods the data were processed using SPSS 26.0 software. The measurement data is expressed by x +/-s, if the data accords with normal distribution and meets the homogeneity of variance, the multi-group comparison adopts one-factor variance analysis, the comparison between every two groups is tested by LSD-t, and the comparison between each administration group and the blank group is tested by Dunnett-t. If the data is distributed in a biased way or the variance homogeneity is not satisfied, a nonparametric test of rank conversion is adopted. The difference is that P is less than 0.05, which has statistical significance

2.1 general conditions and gross morphology of lung tissue in rats compared with those in the sham-operated group, the rats in the model group had lusterless hair, slow response, decreased body temperature, and slow or decreased physical mass growth. Example 1 rats in the high and low dose groups had more hair shine, more responsive response, and recovery of body temperature and body mass than the model group. The lung tissue of the rat in the sham operation group is light red, and bleeding spots, petechiae and ecchymosis are not seen; the lung tissues of the rats in the model group are pale, and scattered bleeding points, black ecchymosis and surface nodular changes can be seen in the gross morphology; the surfaces of lung tissues on both sides of rats in the high and low dose groups become white, a small amount of punctate bleeding spots and a small amount of ecchymosis can be seen, the middle and rear lobes of the right lung are obvious, and the bleeding spots are reduced compared with those in the model group. See fig. 2.

2.2 influence on pulmonary factor of rats compared with the sham-operated group, the rats in the model group had increased pulmonary mass, decreased body mass and increased pulmonary factor, with statistical differences (all P < 0.01). Compared with the model group, the high and low dose groups of example 1 had increased body mass, decreased lung mass and lung coefficient, and the difference was statistically significant (all P < 0.01). Example 1 comparison between high and low dose groups, the difference in lung coefficients in rats was not statistically significant (P > 0.05). See table 6.

Table 6 comparison of lung mass, body mass and lung coefficient for each group of rats (n =10,

)

note: p < 0.01 compared to sham group; compared to the model group, # # P < 0.01.

2.3 compared with the false operation group, the influence of the content of KL-6, IL-6 and IL-21 in the serum of the rat is increased, and the difference is statistically significant (P is less than 0.01). Compared with the model group, the content of KL-6, IL-6 and IL-21 in the high and low dose groups in example 1 is reduced to different degrees, and the difference has statistical significance (P is less than 0.01). Example 1 KL-6 was reduced in the high dose group compared to the low dose group, with a statistically significant difference (P < 0.01). See table 7.

Table 7 comparison of serum KL-6, IL-21 for each group of rats (n =10,

)

note: p < 0.01 compared to sham group; compared with the model group, # # P is less than 0.01; compared to the high dose group, Δ P < 0.01.

2.4 Effect on TGF-beta 1 content in rat Lung tissue (see FIG. 3) compared with the sham-operated group, the model group showed statistically significant differences in TGF-beta 1 content in rat lung tissue (P < 0.01). Compared with the model group, the lung tissue TGF-beta 1 content of rats in the high and low dose groups of the example 1 is reduced to different degrees, and the difference has statistical significance (P is less than 0.01). Example 1 comparison between high and low dose groups, TGF-. Beta.1 content differences were not statistically significant (P > 0.05). See table 8.

Table 8 effect of TGF- β 1 in rat lung tissue groups (n =10,

)

2.5 HE staining results of rat lung tissues of each group show that the rat lung tissue structure of the sham operation group is normal, obvious inflammatory cell exudation and bleeding are not observed in the HE staining, after the model group is molded by bleomycin, the section lesion of the model group is unevenly distributed, the lung tissue structure of the lesion is damaged, alveolar cavities are irregularly collapsed and fused, the alveolar intervals are obviously widened, and inflammatory cell infiltration and fibroblast proliferation are observed inside. The intima hyperplasia of the pulmonary artery and the thickening of the middle layer are achieved, and the thickness ratio of the intima in the artery of the model group is twice as much as that of the artery of the false operation group. Example 1 the pulmonary alveolar structure change, inflammatory cell infiltration and collagen deposition in the high and low dose groups of rats were improved as compared with the model group, and the vasodilation phenomenon was reduced as compared with the model group, as shown in fig. 4.

2.6 the influence of the protein expression of KL-6, TGF-beta 1 and IL-6 in pulmonary tissues of rats with pulmonary fibrosis is compared with that of a sham operation group, and the protein expression of KL-6, TGF-beta 1 and IL-6 in pulmonary tissues of rats in a model group is increased. Compared with the model group, the expression of KL-6, TGF-beta 1 and IL-6 proteins of the high and low dose groups of the embodiment 1 is reduced. See fig. 5.

Example 10 animal experiment 2

Effect of different components on bleomycin-induced pulmonary fibrosis of rats

1 materials and methods (see example 9)

1.1 materials

1.1.1 animals SPF grade male SD rats 30 animals, with a body mass (200 + -20) g, purchased from Kyoho Cawens laboratory animals, inc. Animal production license number: SCXK (threo) 2016-0010. The animal experiment center of the transformation medicine research institute of Shanghai university is bred, and the experimental animals use license numbers: SYXK (Shanghai) 2014-0016. The feeding conditions comprise room temperature of 20-25 ℃, humidity of 40-70%, standard feed, free diet of animals and drinking water. SD rats were housed in pathogen-free SPF-grade animal chambers for 1 week.

1.2 grouping and modeling and intervention SD rats were randomly divided into 3 groups by the random number table method, i.e., the present group (using the composition of example 2), the control group 1 (using the composition of example 6), the control group 2 (using the composition of example 7), 10 per group. The administration method is converted from an equivalent dose ratio table converted from body surface area of human and animal, and the dosage of the rat is converted from clinical dose. The group of the invention was administered at a dose of 14.19g/kg body mass, formulated to a concentration of 1.42g/mL. The present group was administered daily to each concentration example 2 at a high dose and was gavaged at 10mL/kg body mass. The control group 1 was administered at a dose of 14.19g/kg body mass and was prepared at a concentration of 1.42g/mL. The control group 1 was administered with the respective concentrations of example 6 at a daily dose of 10mL/kg body mass by intragastric administration. Control group 2 was administered at a dose of 14.19g/kg body mass and was formulated to have a concentration of 1.42g/mL. Control 2 was administered daily to each concentration example 7 at a high dose and was gavaged at 10mL/kg body mass. The intervention was started on day 1 after the molding of each group until the end of the experiment.

1.3 detection index and method

Expression of KL-6, IL-6 and IL-12 proteins of lung tissues is detected by a Western Blot method. Buckling the prepared SDS-PAGE gel plate on an electrophoresis tank, adding 1 × Running Buffer, pulling out a comb, adding a protein Marker and a sample, 80V,30min;120V,70min electrophoresis. The film transfer tank was placed in an ice-water mixture at 300mA for film transfer at 1h. After blocking with 5% skim milk, an antibody dilution containing the antibody of interest was added and incubated overnight at 4 ℃. Washing the membrane for 5min on a high-low shaking table for the next day by TBST for 3 times; adding an antibody diluent containing a second antibody, and incubating at room temperature for 1-2 h; and finally, preparing A, B liquid of the ECL color developing liquid according to the ratio of 1: 1, and coating the ECL color developing liquid in a tin foil paper to prevent light. The membrane was placed on a detection plate, the residual TBST was carefully aspirated, about 200. Mu.L of developing solution was added, and the membrane was wiped. The secondary antibody was incubated for 2h at room temperature every other day, followed by luminescence photographing with a gel imager and analysis of the band gray values with Image J.

2.1 Effect on rat serum KL-6, IL-21 levels is shown in Table 9

Table 9 comparison of serum KL-6, IL-21 for each group of rats (n =10,

)

rat serum KL-6 content: the control group 1 and the control group 2 are higher than the group of the invention; rat serum IL-6 content: the control group 1 and the control group 2 are higher than the group of the invention; rat serum IL-21 content: the control group 1 and the control group 2 were higher than the group of the present invention. The specific component proportion technical scheme of the invention has obvious effect on treating rat pulmonary fibrosis. The invention reduces the pulmonary fibrosis degree of rats by regulating the expressions of KL-6, IL-6 and IL-21, and the effect of a high-dose group and a low-dose group is better. No rat in each group died, and the medicine safety was good.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. The traditional Chinese medicine composition for treating pulmonary fibrosis diseases is characterized by being prepared from the following raw material medicines in parts by weight: 28-31 parts of radix rehmanniae, 29-32 parts of centella asiatica, 12 parts of curculigo orchioides, 12 parts of epimedium herb, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

2. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw materials in parts by weight: 30 parts of radix rehmanniae, 30 parts of centella, 12 parts of rhizoma curculiginis, 12 parts of herba epimedii, 15 parts of peach kernel, 9 parts of salvia miltiorrhiza, 9 parts of scutellaria baicalensis, 15 parts of dipsacus root and 6 parts of raw liquorice.

3. The use of the Chinese medicinal composition according to any one of claims 1 or 2 in the preparation of a medicament for the treatment of pulmonary fibrosis.

4. The use according to claim 3, wherein the pulmonary fibrotic disease is rheumatoid arthritis-associated pulmonary fibrosis.