Application of sinking ascending decoction in preparation of medicine for improving adriamycin-induced intestinal flora imbalance

Abstract

The invention relates to the field of medicines, in particular to application of sinking ascending decoction in preparation of a medicine for improving adriamycin-induced intestinal flora imbalance. Microbiomics analysis finds that the sinking ascending decoction can improve intestinal flora imbalance induced by doxorubicin. Results of the invention show that the Shuiyang decoction can effectively improve cardiac functions of doxorubicin-induced chronic heart failure rats, alleviate myocardial damage and inhibit myocardial fibrosis and myocardial cell apoptosis, which prompts that the Shuiyang decoction can alleviate cardiotoxicity of doxorubicin and has a protective effect on doxorubicin-induced chronic heart failure.

Description

Technical Field

[0001] The invention relates to the field of medicines, in particular to the application of Shengxian decoction in preparing medicines for improving intestinal flora imbalance induced by adriamycin. Background Art

[0002] Doxorubicin (DOX) is a broad-spectrum anthracycline antitumor drug widely used in the treatment of breast cancer, malignant lymphoma and other cancers. However, the clinical application of DOX is limited by many adverse reactions, among which cardiac toxicity is the most typical, manifested as left ventricular dysfunction and even heart failure. Studies have shown that 26% of cancer patients develop heart failure after receiving DOX treatment. When the cumulative dose reaches 700mg / m 2 The incidence of heart failure in patients receiving DOX chemotherapy can reach 48%, and the cardiotoxicity of DOX is progressive and irreversible, which brings serious health burden to patients. Currently, there is a lack of drugs that can safely and effectively alleviate the cardiotoxicity of DOX in clinical practice, and there is an urgent need to find ideal cardioprotective agents to reduce the cardiotoxicity of DOX.

[0003] Shengxian Decoction comes from the "Medical Records of Chinese and Western Medicine", which was created by the famous Chinese medicine scientist Mr. Zhang Xichun. The prescription includes five medicinal materials: Astragalus, Bupleurum, Cimicifuga, Anemarrhena and Platycodon. It is mainly used to treat various symptoms of "air sinking". It is often used in modern clinical practice to treat coronary heart disease, chronic heart failure and other diseases. Many clinical studies have shown that Shengxian Decoction can significantly improve the cardiac function classification of patients with chronic heart failure, reduce the plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) level, and has good safety.

[0004] However, there are no reports on the application of Shengxian Decoction in alleviating the cardiotoxicity of doxorubicin and improving doxorubicin-induced intestinal flora imbalance. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of Shengxian decoction in preparing a medicine for improving intestinal flora imbalance induced by doxorubicin and reducing the cardiotoxicity of doxorubicin.

[0006] The first aspect of the present invention provides the use of Shengxian Decoction in the preparation of a medicament for improving doxorubicin-induced intestinal flora imbalance.

[0007] Furthermore, Shengxian decoction is used in the preparation of a drug for improving doxorubicin-induced disorders of Proteobacteria and Actinobacteriota.

[0008] Furthermore, Shengxian decoction is used in the preparation of drugs for improving doxorubicin-induced disorders of bacteria such as Muribaculaceae, [Eubacterium]_xylanophilum_group, Bacteroides, Lactobacillus, Allobaculum and Escherichia-Shigella.

[0009] The second aspect of the present invention provides the use of Shengxian Decoction in the preparation of a medicament for alleviating the cardiotoxicity of doxorubicin.

[0010] Furthermore, Shengxian Decoction is used in the preparation of a medicine for improving cardiac function damage caused by doxorubicin.

[0011] Furthermore, Shengxian Decoction is used in the preparation of a drug for improving myocardial injury caused by doxorubicin.

[0012] Furthermore, Shengxian decoction is used in the preparation of drugs for inhibiting myocardial fibrosis and myocardial cell apoptosis induced by doxorubicin.

[0013] Furthermore, Shengxian Decoction is used in the preparation of medicines for treating doxorubicin-induced chronic heart failure.

[0014] The advantages of the present invention are:

[0015] For the first time, the microbiome characteristics and regulated intestinal flora of Shengxian Decoction in regulating doxorubicin-induced chronic heart failure were clarified, which is expected to become a new mechanism of Shengxian Decoction in alleviating doxorubicin-induced chronic heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Effects of Shengxian Decoction on left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in rats with chronic heart failure induced by adriamycin;

[0017] Figure 2 .The effect of Shengxian decoction on serum brain natriuretic peptide (BNP), creatine kinase (CK) and lactate dehydrogenase (LDH) in rats with chronic heart failure induced by adriamycin;

[0018] Figure 3 .Effect of Shengxian Decoction on Beta diversity of intestinal flora in rats induced by adriamycin;

[0019] Figure 4 .Effect of Shengxian Decoction on species composition of intestinal flora at phylum level in rats induced by adriamycin;

[0020] Figure 5 .Effect of Shengxian Decoction on the relative abundance of Proteobacteria and Actinobacteriota in rats induced by adriamycin;

[0021] Figure 6 .The effect of Shengxian decoction on the species composition of intestinal flora at genus level in rats induced by adriamycin; DETAILED DESCRIPTION

[0024] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0025] Example 1: Effect of Shengxian Decoction on cardiac function and intestinal flora in rats with chronic heart failure induced by adriamycin

[0026] 1.1 Experimental instruments and materials

[0027] 2100 small animal ultrasound imager (Visual Sonics, Canada), medical low-temperature refrigerator (Hisense, China), Multiskan1550 full-wavelength multifunctional microplate reader (Thermo Fisher, USA), BK-280 fully automatic biochemical analyzer (BIOBASE, China), 5810R low-temperature high-speed centrifuge (Eppendorf, Germany), 580BR10905 PCR instrument (Bio-rad, USA), NovaSeq6000 desktop sequencer (Illumina, USA), HE-120 electrophoresis instrument (Tanon, China), 2500 gel imager (Tanon, China), 2100 Bioanalyzer (Aglient, USA), 2000 NanoDrop spectrophotometer (Thermo Fisher, USA).

[0028] Doxorubicin hydrochloride for injection (Wanle Pharmaceutical), enalapril maleate tablets (Yangtze River Pharmaceutical), Shengxian Decoction (Pharmaceutical Center of the Second Affiliated Hospital of Naval Medical University), isoflurane (Hebei Yipin Pharmaceutical), depilatory cream (Veting), rat brain natriuretic peptide (BNP) test kit (Jiancheng Biological), rat creatine kinase (CK) detection kit (BIOBASE), rat lactate dehydrogenase (LDH) detection kit (BIOBASE), 5 mL procoagulant tube (BD), MagPure Soil DNA LQ Kit (Magen), Qubit dsDNA assay kit (Life Technologies), Tks Gflex DNA Polymerase (Takara).

[0029] 48 healthy male SPF SD rats, aged 7 to 8 weeks and weighing between 200 and 220 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and kept in the SPF Experimental Animal Center of Naval Medical University. The experimental animal use license number is SYXK (Shanghai) 2020-0009. The experimental animals were kept in a temperature-controlled environment, the room temperature was maintained at 22 ± 2 ° C, and the relative humidity was maintained at 65 ± 5%. The light / dark cycle was 12 h, and the indoor lighting was manually controlled to meet the 12-h diurnal light pattern. The room was well ventilated to ensure the comfort and health of the animals. The animal experiment protocol was approved by the University Animal Ethics Committee.

[0030] 1.2 Experimental methods

[0031] Astragalus (origin Gansu, batch number 2208290133), Bupleurum (origin Shanxi, batch number 2301030093), Platycodon (origin Anhui, batch number 2212160012), Anemarrhena (origin Anhui, batch number 2209160262) and Cimicifuga (origin Liaoning, batch number 21070611) were purchased from Shanghai Cai Tongdetang Chinese Medicine Pieces Co., Ltd. in Wujiang. The preparation of Shengxian Decoction solution (the preparation method refers to Chinese patent document CN115479995B A preferred method for Shengxian Decoction extraction process) was provided by the Special Preparation Center of the Second Affiliated Hospital of Naval Medical University and stored in a 4°C refrigerator for use.

[0032] Preparation of doxorubicin solution: Accurately weigh doxorubicin powder, fully dissolve it in sterile saline, prepare a doxorubicin solution with a concentration of 1.0 mg / mL, sterilize it through a 0.22 μm microporous filter membrane, and use it immediately after preparation.

[0033] Preparation of enalapril solution: Accurately weigh enalapril powder, fully dissolve it with sterile saline, prepare a solution with an enalapril concentration of 0.18 mg / mL, divide it into portions and store it in a refrigerator at 4°C.

[0034] Before use, the above drugs were heated in a 37°C water bath for 10 minutes and shaken thoroughly to prevent precipitation of the drugs.

[0035] 1.3 Experimental animal preparation and grouping

[0036] After one week of adaptive feeding, SD rats were randomly divided into 6 groups: normal group (CON, n = 6), DOX group (n = 10), positive drug enalapril group (ENP, n = 8), Shengxian decoction (hereinafter referred to as SXT) low-dose group (n = 8), SXT medium-dose group (n = 8), SXT high-dose group (n = 8). Among them, the rats in the latter 5 groups were intraperitoneally injected with DOX (2.5 mg / kg / 5d) for 6 consecutive times, with a cumulative dose of 15 mg / kg, and the rats in the CON group were intraperitoneally injected with an equal amount of normal saline. During the experiment, rats were allowed to eat and drink freely. Rats in each group were gavaged at the following doses: the ENP group was gavaged with enalapril (1.8 mg / kg / d); the SXT low-dose group, medium-dose group, and high-dose group were gavaged with SXT 4 g / kg / d (clinical equivalent dose), 8 g / kg / d (2 times clinical equivalent dose), and 16 g / kg / d (4 times clinical equivalent dose), respectively; the CON group and DOX group were gavaged with an equal amount of normal saline every day until day 39. After the last administration, echocardiography was performed, and before the animals were killed, fresh feces of rats in the CON group, DOX group, and SXT group were collected into sterile cryovials, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator for microbiome analysis.

[0037] 1.4 Extraction and library construction of intestinal bacterial DNA

[0038] 1.4.1 Extraction of intestinal bacterial DNA

[0039] The stool samples collected from the CON group, DOX group, and SXT high-dose group (16 g / kg) were taken out of the -80°C refrigerator and operated according to the instructions of the DNA extraction kit to extract bacterial DNA from the samples. The DNA concentration and integrity were then measured by agarose gel electrophoresis and NanoDrop 2000 spectrophotometer. Using genomic DNA as a template, PCR amplification was performed for the 16S V3-V4 region using primers 343F (5′-TACGGRAGCAGCAG-3′) and 798R (5′-AAGGTATCTAATCCT-3′). In the PCR reaction, specific primers with barcodes and Takara's Tks Gflex DNA Polymerase were used to ensure amplification efficiency and accuracy. The amplification system was 2×Gflex PCR Buffer 15 μL, 5 pmol / μl primer F 1 μL, 5 pmol / μl primer R 1 μL, Template DNA 1 μL, Tks Gflex DNA Polymerase (1.25 U / μL) 0.6 μL, and water was added to 30 μL. The amplification conditions are shown in Table 1.

[0040] Table 1 Conditions for one round of PCR amplification

[0041]

[0042] 1.4.2 PCR amplification and library construction

[0043] The first-round PCR product was first detected by electrophoresis and purified by magnetic beads, and then amplified as a template for the second-round PCR. The amplification system was 2×Gflex PCR Buffer 15μL, Tks Gflex DNA Polymerase (1.25U / μL) 0.6μL, Adapter I5 1μL, Adapter I7 1μL, the first-round product 50ng, and water was added to 30μL. The amplification conditions are shown in Table 2. The amplified product was again detected by electrophoresis and purified by magnetic beads, and then quantified using the Qubit dsDNA assay kit. Equal amounts were mixed according to the concentration of the PCR product, and then sequenced and analyzed on the machine.

[0044] Table 2 Second round PCR amplification conditions

[0045]

[0046] 1.5 Bioinformatics Analysis Process

[0047] The original data was stored in FASTQ format. The data processing process first used Cutadapt software to cut the original sequence and remove the primer sequence. Subsequently, the DADA2 software was used to perform quality control steps such as quality filtering, noise reduction, splicing, and chimera removal on the double-end raw data after preliminary processing, and analyzed according to the default parameters of QIIME 2 (2020.11) to obtain representative sequences and ASV (Amplicon Sequence Variant) abundance tables. Then, the QIIME 2 software package was used to screen the representative sequences of each ASV and compare them with the Silva (version138) database. The species alignment annotation was analyzed using the default parameters in the q2-feature-classifier software package. Then, bioinformatics analysis was carried out based on the Ouyi Cloud platform, including diversity analysis, species composition analysis, species difference analysis, and KEGG function prediction analysis.

[0048] 1.6 Results and Discussion

[0049] 1.6.1 Shengxian Decoction can improve chronic heart failure induced by adriamycin

[0050] Echocardiography was used to evaluate the cardiac function of the rats in each group. Figure 1As shown in the figure, compared with the CON group, the cardiac ultrasound indexes of rats in the DOX group changed significantly, as shown by a significant decrease in LVEF and LVFS values ​​(P<0.001); compared with the DOX group, the LVEF and LVFS values ​​of rats in the ENP group and the three doses of SXT groups increased significantly (P<0.01). The echocardiographic results showed that SXT could significantly improve the cardiac function damage caused by DOX and had a protective effect on DOX-induced chronic heart failure.

[0051] like Figure 2 As shown in the figure, compared with the CON group, the level of serum heart failure marker brain natriuretic peptide (BNP) in the DOX group was significantly increased (P<0.001), while the serum BNP levels in the ENP group and the low-, medium-, and high-dose SXT groups were significantly lower than those in the DOX group (P<0.001). Figure 2 As shown in BC, by comparing the serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels of rats in each group, it was found that compared with the CON group, the CK and LDH values ​​of rats in the DOX group were significantly increased (P<0.001), while the serum CK and LDH values ​​of rats in the ENP group and the low-, medium-, and high-dose SXT groups were significantly lower than those in the DOX group (P<0.001). The above results indicate that SXT can significantly inhibit DOX-induced myocardial damage in rats with chronic heart failure.

[0052] 1.6.2 Shengxian Decoction can improve doxorubicin-induced intestinal flora imbalance

[0053] Beta diversity analysis can be used to assess the similarity or difference in bacterial composition between groups. Figure 3 As shown in the figure, principal coordinate analysis (PCoA) based on the Bray-Curtis distance matrix found that the DOX group was significantly separated from the CON group, indicating that the microbiota composition of the DOX group was significantly different from that of the CON group, while the SXT group tended to be away from the DOX group and close to the CON group, indicating that the microbiota composition of the SXT group was similar to that of the CON group, and SXT could alleviate the intestinal microbiota imbalance caused by DOX.

[0054] In order to understand the bacterial community composition structure of each group of rat feces samples, we drew the relative abundance bar graphs of the community structure of each group of rat feces at the phylum and genus classification levels. Figure 4 As shown in the figure, at the phylum level, the dominant bacterial phyla of each group of rats were mainly from Bacteroidetes, Firmicutes, Desulfobacterota and Proteobacteria, and their total relative abundance in each group of samples was >98%; Figure 5As shown in the figure, compared with the CON group, the relative abundance of Proteobacteria and Actinobacteriota in the DOX group increased significantly (P<0.01), while the relative abundance of Proteobacteria and Actinobacteriota in the SXT group was significantly lower than that in the DOX group (P<0.01). The significant increase in the abundance of Proteobacteria is an important feature of dysbiosis or unstable intestinal microbial community structure, and studies have found that DOX can cause a significant increase in Proteobacteria in rat feces. The above results indicate that SXT can significantly inhibit the dysbiosis of Proteobacteria and Actinobacteriota induced by DOX.

[0055] like Figure 6 As shown in the results, compared with the CON group, the relative abundance of Muribaculaceae and [Eubacterium]_xylanophilum_group in the DOX group was significantly decreased (P<0.01), and the relative abundance of Bacteroides, Lactobacillus, Allobaculum and Escherichia-Shigella was significantly increased (P<0.05). Compared with the DOX group, the relative abundance of Muribaculaceae and [Eubacterium]_xylanophilum_group in the SXT group was significantly increased (P<0.05), and the relative abundance of Bacteroides, Lactobacillus, Allobaculum and Escherichia-Shigella was significantly decreased (P<0.01). The above results indicate that SXT can significantly inhibit the DOX-induced imbalance of Muribaculaceae, [Eubacterium]_xylanophilum_group, Bacteroides, Lactobacillus, Allobaculum and Escherichia-Shigella.

[0056] 1.6.3 Discussion

[0057] The present invention constructs a DOX-induced chronic heart failure rat model to conduct a pharmacodynamic evaluation of SXT in improving DOX-induced chronic heart failure. The results show that compared with the CON group, the weight gain of rats in the DOX group was slow, the cardiac ultrasound indicators LVEF and LVFS values ​​decreased significantly, and the serum heart failure marker BNP level and myocardial enzyme spectrum level (CK, LDH) increased significantly. Angiotensin-converting enzyme inhibitor ENP, as a drug recommended for use in the Guidelines for the Prevention and Treatment of Anthracycline Cardiotoxicity (2020 Edition) and the National Guidelines for Heart Failure (2023), plays an important role in the prevention and treatment of anthracycline cardiotoxicity and heart failure. Therefore, the present invention selects ENP as a positive drug to evaluate the protective effect of SXT on DOX-induced chronic heart failure. The results showed that compared with the DOX group, the ENP group and different doses of SXT groups could significantly improve the slow weight gain of DOX-induced chronic heart failure rats, restore LVEF and LVFS values, and significantly inhibit the increase of serum BNP, CK, and LDH levels. At the same time, they restored the myocardial tissue structure, reduced the area of ​​myocardial fibrosis, and alleviated myocardial cell apoptosis. Among the SXT groups, the high-dose group had the most significant improvement effect on multiple indicators such as rat LVEF, LVFS, body weight, and serum BNP.

[0058] Microbiome results showed that SXT could improve the diversity of intestinal flora in rats induced by DOX and significantly reverse the changes in intestinal flora structure induced by DOX. Through species difference analysis, it was found that DOX caused a significant increase in the relative abundance of multiple opportunistic pathogens (such as Escherichia-Shigella) and a significant decrease in the relative abundance of probiotics (such as Muribaculaceae). Existing literature has shown that Muribaculaceae is a probiotic that plays an important role in the metabolism of sugars and polyphenols and helps to produce short-chain fatty acids such as propionic acid. Its relative abundance is negatively correlated with inflammatory response; Escherichia-Shigella is an opportunistic pathogen with clear functions. Its abundance in patients with heart failure is significantly increased, and the proliferation of Escherichia-Shigella will release toxic substances such as lipopolysaccharide, leading to an increase in proinflammatory factors in the body and aggravation of cardiac function damage.

[0059] The above results suggest that SXT can effectively improve the cardiac function of rats with chronic heart failure induced by DOX, reduce myocardial damage, inhibit myocardial fibrosis and myocardial cell apoptosis, indicating that SXT can reduce the cardiotoxicity of DOX and has a protective effect on chronic heart failure induced by DOX, among which the high-dose group has the best effect. Microbiome analysis found that Shengxian Decoction can improve DOX-induced intestinal flora imbalance.

[0060] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of Shengxian Decoction in the preparation of drugs for improving doxorubicin-induced intestinal flora imbalance.

2. The use according to claim 1, characterized in that: Application of Shengxian decoction in the preparation of medicines for improving doxorubicin-induced disorders of Proteobacteria and Actinobacteriota.

3. The use according to claim 1, characterized in that: The application of Shengxian decoction in the preparation of medicines for improving doxorubicin-induced disorders of bacteria such as Muribaculaceae, [Eubacterium]_xylanophilum_group, Bacteroides, Lactobacillus, Allobaculum and Escherichia-Shigella.

4. Application of Shengxian Decoction in the preparation of drugs for reducing the cardiotoxicity of doxorubicin.

5. The use according to claim 4, characterized in that: Application of Shengxian Decoction in the preparation of medicines for improving cardiac function damage induced by doxorubicin.

6. The use according to claim 4, characterized in that: Application of Shengxian Decoction in the preparation of medicines for improving myocardial injury induced by doxorubicin.

7. The use according to claim 4, characterized in that: Application of Shengxian decoction in the preparation of drugs for inhibiting myocardial fibrosis and myocardial cell apoptosis induced by doxorubicin.

8. The use according to claim 4, characterized in that: Application of Shengxian Decoction in the preparation of medicines for treating doxorubicin-induced chronic heart failure.

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