Traditional Chinese medicine composition and application

Abstract

The invention provides a traditional Chinese medicine composition and application, and relates to the technical field of medical biologica.In the embodiment of the invention, the treatment effect of the traditional Chinese medicine composition comprising radix astragali, radix et rhizoma rhei, leech, radix salviae miltiorrhizae and herba epimedii on DN model rats is studied through transcriptome sequencing. The rat models are divided into a control group, a DN group and a treatment group. Creatinine (Cr), blood urea nitrogen (BUN), and urinary albumin levels of each group are measured. And the pathological change of the kidney tissue is observed by adopting Masson dyeing and PAS dyeing. The result shows that compared with the DN group, the Cr, BUN and urinary albumin levels of the treatment group are obviously reduced (plt; 0.05) of the method. And the masson dyeing shows that the kidney collagen fibers in the treatment group are reduced. In addition, PAS staining shows that the mesangial expansion of the treatment group is less, and the extracellular matrix deposition is reduced. It is indicated that the composition can effectively reduce the Cr, BUN and urinary albumin levels of DN rats, and has a certain degree of kidney protection effect.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of pharmaceutical biotechnology, and in particular to a Chinese medicine composition and application thereof. Background Art

[0002] Diabetic nephropathy (DN) is one of the most serious chronic microvascular complications of diabetes, characterized by persistent proteinuria and progressive hypertension. Relevant data show that up to half of DN patients may develop end-stage renal disease (ESRD), and due to the prevalence of diabetes, DN has become one of the main causes of ESRD worldwide. DN has a hidden onset and early symptoms are not obvious, which makes diagnosis based on clinical manifestations alone complicated.

[0003] Current conventional interventions for DN include body fat and weight management, smoking cessation, and regulation of blood sugar and blood pressure. However, due to differences among DN patients and compliance issues, effective results cannot be uniformly produced.

[0004] Clinically, proteinuria is usually controlled by inhibiting the renin-angiotensin-aldosterone system (RAAS); however, this approach is not suitable for patients with DN without hypertension. Although more extensive blockade of RAAS is theoretically advantageous, the combined use of RAAS inhibitors leads to an increased risk of acute kidney injury (AKI) and hyperkalemia. Although sodium-glucose cotransporter 2 (SGLT2) inhibitors have been used clinically, patients still face a significant risk of progression to ESRD due to various pathophysiological disorders in DN. However, it will take a considerable amount of time to develop new targeted therapies, especially for glomerular hyperfiltration, inflammation, and fibrosis. Therefore, a new treatment option for diabetic nephropathy is urgently needed. Summary of the invention

[0005] The embodiment of the present invention provides a Chinese medicine composition and application, so as to provide a solution for treating diabetic nephropathy.

[0006] A first aspect of an embodiment of the present invention provides a traditional Chinese medicine composition, comprising: astragalus, rhubarb, leech, salvia miltiorrhiza and epimedium.

[0007] Optionally, the Chinese medicine composition is in the form of powder, aqueous solution, tablet or capsule.

[0008] The second aspect of the embodiment of the present invention provides the use of the traditional Chinese medicine composition described in the first aspect in the preparation of a drug for treating diabetic nephropathy, wherein the traditional Chinese medicine composition acts on the targets: TIMP1, LOX, POSTN, NFKBIA and SHH, and the traditional Chinese medicine composition is used to reduce ECM deposition and alleviate renal fibrosis to slow down the development of diabetic nephropathy.

[0009] Optionally, the Chinese medicine composition inhibits TIMP1 expression, reduces the inhibition of MMP9, and promotes the degradation of ECM.

[0010] Optionally, the Chinese medicine composition inhibits the transformation of renal epithelial cells from epithelial cells to mesenchymal cells by inhibiting the expression of LOX, thereby reducing the occurrence of renal fibrosis.

[0011] Optionally, the Chinese medicine composition inhibits the expression of POSTN, slows down renal tubular cell fibrosis, slows down the development of diabetic nephropathy, blocks the mutual reinforcement between TGF-β and POSTN, and slows down the progression of diabetic kidney fibrosis.

[0012] Optionally, the Chinese medicine composition achieves kidney protection by inhibiting the expression of NFKBIA, activating other alternative anti-inflammatory signaling pathways, and compensating for the regulation of the NF-κB pathway.

[0013] Optionally, the Chinese medicine composition inhibits SHH expression to reduce ECM synthesis, and the Chinese medicine composition inhibits the role of SHH signaling pathway in the progression of renal fibrosis by inhibiting SHH expression.

[0014] The third aspect of the embodiment of the present invention provides the use of the traditional Chinese medicine composition described in the first aspect in the preparation of a drug for reducing ECM deposition, wherein the traditional Chinese medicine composition inhibits TIMP1 expression, reduces the inhibition of MMP9, and promotes the degradation of ECM; the traditional Chinese medicine composition inhibits SHH expression to reduce the synthesis of ECM.

[0015] The third aspect of the embodiment of the present invention provides the use of the traditional Chinese medicine composition described in the first aspect in the preparation of a drug for alleviating renal fibrosis, wherein the traditional Chinese medicine composition inhibits the transformation of renal epithelial cells from epithelial cells to interstitial cells by inhibiting the expression of LOX, thereby alleviating the occurrence of renal fibrosis; the traditional Chinese medicine composition inhibits the expression of POSTN, thereby slowing down renal tubular cell fibrosis, slowing down the development of diabetic nephropathy, and blocking the mutual reinforcement between TGF-β and POSTN to slow down the progression of diabetic renal fibrosis; the traditional Chinese medicine composition inhibits the expression of NFKBIA, activates other alternative anti-inflammatory signaling pathways, and compensates for the regulation of the NF-κB pathway to achieve a renal protective effect; the traditional Chinese medicine composition inhibits the expression of SHH, thereby inhibiting the role of the SHH signaling pathway in the progression of renal fibrosis.

[0016] In the embodiment of the present invention, the therapeutic effect of a Chinese medicine composition including Astragalus, Rhubarb, Leech, Salvia miltiorrhiza and Epimedium on DN model rats was studied by transcriptome sequencing. The rat model was divided into a control group, a DN group and a treatment group. The levels of creatinine (Cr), blood urea nitrogen (BUN) and urinary albumin in each group were measured. Masson staining and PAS staining were used to observe the pathological changes of renal tissue. The renal tissue samples of each group of rats were taken for RNA sequencing (RNA-seq) and quality control. Differentially expressed genes (DEGs1 and DEGs2) were identified by differential expression analysis, and their functions were explored by functional enrichment analysis. DEGs related to the treatment of the composition were obtained by the merging process of reversely expressed DEGs1 and DEGs2, followed by functional analysis, and key targets were selected using a protein-protein interaction (PPI) network. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed on each group to evaluate the expression of key targets. Compared with the DN group, the levels of Cr, BUN and urinary albumin in the treatment group were significantly reduced (p<0.05). Masson staining showed that renal collagen fibers were reduced in the treatment group. In addition, PAS staining showed less mesangial expansion and reduced extracellular matrix deposition in the treatment group. After QC of RNA-seq data, 620 DEGs1 and 563 DEGs2 were identified and enriched in multiple pathways. DEGs1 up-regulated and DEGs2 down-regulated, DEGs1 down-regulated and DEGs2 up-regulated, resulting in 172 and 39 cross-genes, forming 211 genes related to the treatment of the composition. From the PPI, five key targets were identified: LOX, TIMP1, NFKBIA, POSTN and SHH. It is worth noting that RT-qPCR results showed that the expression levels of TIMP1, NFKBIA and SHH in the DN group were increased compared with the control group, while the expression levels of TIMP1, NFKBIA and SHH in the treatment group were decreased compared with the DN group. The composition can effectively reduce the levels of Cr, BUN and urinary albumin in DN rats, showing a certain degree of renal protection.

[0017] The Chinese medicine composition proposed in the embodiment of the present invention can be used in combination with Western medicine to improve the therapeutic effect of diabetic nephropathy in multiple ways, thereby enhancing the overall benefit of the treatment. The Chinese medicine composition not only shows certain advantages in terms of efficacy, but also has a relatively low price, which can reduce the economic burden on patients. The Chinese medicine composition can be further prepared into powders, aqueous solutions, tablets or capsules, and the method of taking is simple, does not require complicated operations or special equipment, and allows patients to easily integrate into their daily lives, thereby ensuring the continuity and convenience of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0019] Figure 1 It is a statistical result diagram of the control group (Control), DN group and treatment group (YSXZF) provided in the embodiment of the present invention; Figure 2 It is a Masson staining result diagram and collagen fiber area statistical diagram of three groups of rats provided in an embodiment of the present invention; Figure 3 The PAS staining result diagram, the extracellular matrix statistical diagram and the glomerular injury statistical diagram of three groups of rats provided in the embodiments of the present invention; Figure 4 is a schematic diagram of QC results provided by an embodiment of the present invention; Figure 5 It is a schematic diagram of the expression level results of LOX, TIMP1, NFKBIA, POSTN and SHH of three groups of rats provided in the examples of the present invention. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] There is currently no specific drug for the treatment of diabetic nephropathy in clinical practice. Currently, during the diagnosis and treatment process, clinicians use ACEI and ARB, but diabetic nephropathy still has a great risk of progressing to end-stage renal disease. Aldosterone receptor antagonists are another type of RAAS inhibitors. The most commonly used aldosterone receptor antagonist in clinical practice is spironolactone, but it has a higher risk of causing hyperkalemia. In addition, RAAS inhibitors are not suitable for diabetic patients without hypertension. Advanced glycation end products (AGEs) are a type of product of rapid non-enzymatic reactions of aldehydes or ketones with protein substances. Their binding to receptors can promote oxidative stress and the release of inflammatory mediators such as IL-6 and TGF-β1. It is an important initiating factor for the onset of diabetic nephropathy, but currently AGEs pathway-related drugs have not been put into clinical use. Epalrestat is the first aldose reductase inhibitor to enter clinical practice. It is currently mainly used to improve diabetic neuropathy, and there are no clinical trials to explore its renal protective effects. For new targeted drugs against glomerular hyperfiltration inflammation and fibrosis, the drugs that currently show promise include rubusta (a selective inhibitor of protein kinase C (PKC) β), baricitinib (a selective JAK1 and JAK2 inhibitor), pentoxifylline (an anti-inflammatory and anti-fibrotic agent), and atrasentan (a selective endothelin A receptor antagonist). However, so far, the above drugs have not been used to treat diabetic nephropathy.

[0022] Based on this, in the embodiments of the present invention, a Chinese medicine composition and its application are proposed through a series of research and exploration to treat diabetic nephropathy.

[0023] The Chinese medicine composition provided by the embodiment of the present invention comprises: astragalus, rhubarb, leech, salvia miltiorrhiza and epimedium.

[0024] Among them, Astragalus can protect renal function and reduce proteinuria in patients with kidney disease, but its exact molecular details are still insufficient. Palm rhubarb has the effects of reducing proteinuria, lowering blood sugar and improving renal function in patients with diabetic nephropathy. Salvia miltiorrhiza can regulate renal hemodynamics, oxidative metabolism and inflammation, thereby avoiding various renal cell injuries. Traditional Chinese medicine preparations containing Epimedium and Astragalus combined with conventional Western medicine can reduce 24-hour urine protein. Leeches have excellent anti-thrombotic effects. Although the various components in this traditional Chinese medicine composition have potential for treating diabetic nephropathy, their complex interactions and multi-target effects need to be more comprehensively explored beyond a few applied individuals.

[0025] Transcriptomics involves the study of RNA molecules transcribed in cells or tissues at a specific time, focusing mainly on gene expression patterns and their regulatory mechanisms. Transcriptomics is widely used in the study of traditional Chinese medicine, disease mechanisms, and biological pathways, such as the potential mechanisms of Chinese medicine in improving diseases such as acute lung injury, non-alcoholic fatty liver disease, and asthma. In an embodiment of the present invention, the potential effects of a Chinese medicine composition on DN were studied by transcriptomics. Further bioinformatics analysis revealed the key regulatory targets of the Chinese medicine composition in DN. Reverse transcription quantitative polymerase chain reaction (RT-PCR) was then used to verify the expression of these targets in DN renal tissue after treatment with the Chinese medicine composition. It aims to provide new evidence for the treatment of DN and provide a new theoretical basis for clinical relief of DN progression.

[0026] Specifically, in the present embodiment, healthy male Wistar rats (6-8 weeks old, initial weight 180-220 g) were selected for 1 week of adaptive feeding, and then randomly divided into a control group, a DN group, and a treatment group (fed with the Chinese medicine composition YSXZF in the present embodiment), with 5 rats in each group. The clinical formula and dosage of the Chinese medicine composition (YSXZF) are shown in Table 1.

[0027] Table 1 Ingredients and dosage of Chinese medicine composition

[0028] The control group was fed with a normal diet, while the DN group and the treatment group were fed with a high-sugar and high-fat diet. After fasting for 16 hours at night, the 10 model rats were given a single intraperitoneal injection of streptozotocin (STZ) 60 mg / kg to establish a diabetes model, and physical signs were monitored weekly. Blood glucose levels >16.7 mmol / L indicated successful diabetes modeling. The DN group and the treatment group continued to be fed with a high-sugar and high-fat diet for 14 days, and 24-hour urine samples were collected. Urine protein quantification ≥30 mg confirmed that the DN model was successfully established. After the modeling was completed, the treatment group was given a dose of 1.67 g / kg / d of the Chinese medicine composition every day, and the control group and the DN group were given an equal amount of distilled water at the same time every day. During the three-month treatment period after successful modeling, physical signs were monitored weekly.

[0029] After 12 weeks, the rat eyeballs were removed, whole blood was collected, and after standing for 2 hours, it was centrifuged at 3000 rpm for 30 minutes at 4°C. The supernatant was collected and the levels of creatinine and urea nitrogen were determined. The rats were then anesthetized with 1% sodium pentobarbital (80 mg / kg) and euthanized by cervical dislocation. Serum and diseased kidney tissues from the control group, DN group, and treatment group were collected, weighed, and analyzed. Half of the left kidney was fixed in 4% paraformaldehyde for Masson staining and PAS staining. 1 / 4 of the left kidney was stored at -80°C for PCR analysis, and the other 1 / 4 was stored in 4°C RNA protection solution overnight and then transferred to -80°C for transcriptome sequencing. The right kidney was temporarily stored in 4% paraformaldehyde.

[0030] In the present embodiment, creatinine and urea nitrogen levels were measured by enzyme-linked immunosorbent assay (ELISA).

[0031] Specifically, the determination of creatinine (Cr) includes: filling 50 μL of different concentrations (Cr: 24, 12, 6, 3, 1.5, 0 μmol / L) of standard substances in each standard well. First, add 25 μL of sample diluent and then 25 μL of the sample to be tested to the sample well on the ELISA plate. Add 100 μL of enzyme-labeled reagent to each well. Seal the plate with a sealing film and incubate at 37°C for 60 minutes. After washing, add the substrate solution and measure the absorbance (optical density (OD) value) of each well at a wavelength of 450 nm.

[0032] The determination of blood urea nitrogen (BUN) includes: After diluting the standard, accurately add 50 μL of each standard solution to the designated wells on the ELISA plate. First add 25 μL of sample diluent to the sample well to be tested, and then add 25 μL of the sample to be tested (the dilution factor is 2 times). Incubate the culture dish for 30 minutes and then wash. Add 50 μL of enzyme label reagent to each well, incubate for 30 minutes and then wash. Finally, add 50 μL of substrate solution A to each well, and then add 50 μL of substrate solution b. After 10 minutes, add 50 μL of stop solution to terminate the reaction. Measure the OD value of each well within 15 minutes at a wavelength of 450nm.

[0033] In an embodiment of the present invention, the specific method of Masson staining includes: soaking the collected rat kidney tissue in 4% paraformaldehyde, fixing it for 24-48 hours, dehydrating, clearing, wax impregnation, embedding, and slicing it into 3μm thick slices. Before staining, A1 and A2 are mixed in a ratio of 1:1 to prepare Weigent hematoxylin iron staining solution, and the slices are covered and stained for 5-10 minutes. The Masson bleaching solution is used for 3-5 minutes, and then rinsed with distilled water for 30 seconds. Then stain with Lichunhong Fuchsin solution for 5-10 minutes. During the process, a weak acid working solution is prepared with a ratio of distilled water to a weak acid solution of 2:1, and allowed to stand for 30 seconds. After removing excess liquid, a phosphoric acid molar acid solution is applied for 1-2 minutes, and then an aniline blue staining solution is applied for 1-2 minutes, followed by rinsing with a weak acid working solution for 30 seconds. Finally, 95% ethanol is used for rapid dehydration for 2-3 seconds, followed by two rounds of absolute ethanol dehydration, each lasting 5-10 seconds. The sections were then washed twice with xylene for 1–2 min each and sealed with neutral glue for section scanning.

[0034] In an embodiment of the present invention, the specific method of PAS staining includes: fixing the collected rat kidney tissue with a 4% paraformaldehyde solution, embedding in paraffin, and slicing. The tissue slices were baked in a 64°C incubator for 1 hour and then dewaxed in xylene for 20 minutes. Each slice was hydrated by 100% ethanol I (5 minutes) → 100% ethanol II (5 minutes) → 95% ethanol (5 minutes) → 80% ethanol (3 minutes) → 70% ethanol (2 minutes). Then washed three times in PBS for 5 minutes each time. The periodic acid solution was balanced to room temperature and 100 μl of solution was added to each sample. The sample was incubated in a light-proof humidified chamber for 10 minutes. After removing the periodic acid solution, the slices were immersed in distilled water and washed on a shaker for 5 minutes. Then 100 μl of Schiff's reagent was added to each sample and incubated in a light-proof humidified chamber at 37°C for 30 minutes to 1 hour. Differentiate with hydrochloric acid-ethanol solution for 30 seconds, then rinse with tap water twice, each time for 3 seconds. Finally, the sections were dehydrated, cleaned, and mounted for scanning.

[0035] In the embodiment of the present invention, RNA extraction and detection were performed using the following specific method: RNA was extracted from the left kidney tissue of DN using TRIzol (Invitrogen, CA, USA) and following the protocol. The purity and concentration of the isolated RNA were detected using a ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). In order to ensure the integrity of the RNA, a bioanalyzer 2100 (Agilent, CA, USA) was used and verified by agarose electrophoresis. The total RNA required for further analysis was greater than 1 μg.

[0036] In the embodiment of the present invention, the following specific method is used for cDNA library construction and sequencing: Invitrogen SuperScript™ II reverse transcriptase (No. 1896649) is used to synthesize the initial cDNA chain. Subsequently, RNase H (NEB, product number m0297) and Escherichia coli DNA polymerase I (NEB, product number m0209) are used to synthesize the second cDNA chain. In order to repair the cDNA ends, dUTP solution (Thermo Fisher, product number R0133) is used. Subsequently, after adding the a tail, the second chain is subjected to magnetic bead screening and purification. The second chain is digested with UDG enzyme (NEB, product number m0280). The cDNA library is sequenced on the Illumina Novaseq™ 6000 double-end PE150 platform. The mRNA library is prepared using the TruSeq small RNA sample preparation kit and sequenced on the Illumina Hiseq2500 of LC-BIO, following the standardized method.

[0037] In an embodiment of the present invention, the specific method of RNA-seq data preprocessing includes: in order to determine the quality of RNA-seq, the original fastq data of the transcriptome is processed using Fastqc, and the quality control (QC) results are summarized using multiqc. Subsequently, the base distribution in the RNA-seq data is checked for AT and GC separation. Sequence comparison is performed with reference to the rat genome creation index. HISAT2 is used for comparison, and samtools is used to convert the sam file into a bam file. The position distribution of Reas on different chromosomes is also statistically analyzed. The number of MappedReads in different regions (exons, introns) of the specified reference genome is counted. Then, featucounts with count matrix transformation is used to quantitatively analyze the bam file. The number of MappedReads and the length of the transcript in the sample are normalized by the FPKM method as an indicator of the target expression level or transcript.

[0038] In the embodiment of the present invention, the specific method of identifying differentially expressed genes and gene ontology (GO) enrichment analysis by differential expression analysis includes: using the software 'DESeq2' version 1.34.0 to analyze RNA sequencing data and identify differentially expressed genes (DEGs) between the control group, DN group (DEG1) and treatment group (DEG2). Based on the actual data set, the screening criteria were adjusted, the P value was less than 0.05, and the log2FoldChange absolute value was greater than 1. In order to explore the functional effects and pathways associated with these DEGs, the "ClusterProfiler" software package, version 4.6.0, was used to perform KEGG and GO enrichment analysis on each group of DEGs, and the significance threshold P was less than 0.05.

[0039] In the embodiment of the present invention, the specific steps of identification and functional analysis of genes related to the treatment of Chinese medicine composition include: In order to obtain DEGs that play a role in the occurrence and treatment of DN, in the embodiment of the present invention, DEGs1 up-regulated and DEGs2 down-regulated are respectively used as intersection points, DEGs1 down-regulated and DEGs2 up-regulated are respectively used as intersection points, and the intersection genes of these two parts are connected in series to obtain DEGs related to the treatment of Chinese medicine composition. Spearman correlation analysis was used to analyze the inter-gene correlation of DEGs related to the treatment of Chinese medicine composition, and the threshold was correlation coefficient (cor)>0.3, P<0.05. In order to better explain the potential biological effects of these genes, GO and KEGG pathway enrichment analysis was performed on DEGs related to the treatment of Chinese medicine composition using the same method as before. The gene fragments related to the treatment of Chinese medicine composition were uploaded to the STRING database (http: / / www / string-db.org / , interaction score>0.4), and the results were imported into 3.7.0 Cytoscape for protein-protein interaction (PPI) network construction. Then, the CytoHubba plug-in was used to score the genes using five algorithms, namely degree, neighborhood component centrality (NCC), eigenvector-pagerank centrality (EPC), and maximum neighborhood component (MNC). The top 10 candidate DEG genes related to the treatment with traditional Chinese medicine composition were retrieved, and the genes that satisfied the five algorithms were screened as key targets.

[0040] In the embodiment of the present invention, the specific method of molecular regulatory network analysis includes: in order to find other genes related to the functions of the five key targets, GeneMANIA (http: / / www.genemania.org / ) is used to predict the five targets related to gene functions and their functions, and the results are presented as a gene-gene interaction (GGI) network. In order to explore the five key targets and the transcription factors (TFs) that regulate genes, the interaction map between TFs and key targets is drawn using Cytoscape software based on the information provided by the JASPAR database (https: / / jaspar.elixir.no / ). The regulatory mechanism of the five targets is studied, and the regulatory network of mrna and mirna is constructed. First, the miRWalk database (http: / / mirwalk.umm.uni-heidelberg.de / ) is used to predict the miRNAs related to the five targets and generate mRNA-miRNA pairs. Then, based on the mirna obtained above, the Cytoscape software is used to perform network positioning on the mRNA-miRNA pairs.

[0041] In the embodiment of the present invention, the specific method for analyzing the expression of key targets includes: In order to evaluate the expression of key targets, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR) on 5 controls, 5 DNs and 5 Chinese medicine composition samples. The RT-qPCR amplification conditions were 40 cycles of 95°C for 1 minute, 95°C for 20 seconds, 55°C for 20 seconds, and 72°C for 30 seconds. The qPCR primers are listed in Table S1. The relative expression of key targets was calculated using the 2-△CT method.

[0042] In the present embodiment, R (version 4.2.2) software was used to process and analyze RNA-seq data. ELISA analysis was statistically analyzed using Prism GraphPad software, and the experimental data were expressed as mean ± standard error. The t-test was used to compare the quantitative data between the groups, and P < 0.05 was considered statistically significant.

[0043] In the present embodiment, the vital signs of the rats were stable throughout the study, demonstrating the safety of the Chinese medicine composition. When comparing the control group, DN group, and treatment group (YSXZF), the present embodiment found that the DN group had significantly higher levels of Cr, BUN, and urine protein than the control group (p<0.05), as expected. In contrast, treatment with the Chinese medicine composition significantly reduced the levels of Cr, BUN, and urine protein compared with the DN group (p<0.05) (e.g. Figure 1 As shown, Figure 1 The statistical results of the control group (Control), DN group and treatment group (YSXZF) are shown, wherein Part A shows the statistical results of the body weight, blood sugar and kidney weight of the three groups of rats, and Part B shows the statistical results of the Cr, BUN and urine protein levels of the three groups of rats after administration of the Chinese medicine composition). Masson staining shows that collagen fibers are blue, and muscle fibers, fibrin and red blood cells are red. Compared with the control group, the collagen fibers in the DN group increased significantly, while the collagen fibers in the treatment group decreased significantly compared with the DN group (as shown in Figure 2). Figure 2 As shown in Figure 2, which shows Masson staining results and collagen fiber area statistics of three groups of rats). PAS staining is used to evaluate glomerular sclerosis of renal tissue, and the percentage of tubulointerstitial damage is scored, including glomerular and tubular atrophy and expansion, cast formation, interstitial mononuclear cell accumulation, and extracellular matrix accumulation (interstitial volume). The scoring system is: 1 = less than 10%, 2 = 10-25%, 3 = 26-50%, 4 = 51-75%, 5 = 76-95%, 6 = more than 95%. Statistical analysis showed that compared with the control group, the DN group had increased mesangial expansion and extracellular matrix deposition, while these parameters were reduced in the treatment group compared with the DN group (as shown in Figure 2). Figure 3As shown, it shows the PAS staining results, extracellular matrix statistics and glomerular injury statistics of the three groups of rats).

[0044] In the embodiments of the present invention, the QC results show that the Phred score is generally around 40% (e.g. Figure 4 As shown in part A, Figure 4 The QC result diagram is shown, where Part A shows the Phred score results, Part B shows the Log10 (FPKM) results, and Part C shows the FPKM expression distribution diagram of a single sample), indicating that the sequencing quality is excellent. At the same time, there is no obvious AT and GC separation in the RNA-seq data. The chromosome position distribution shows that the overall comparison is good, and there is no obvious gap region in the chromosome comparison. The overall distribution range of gene expression is 0 ~ 2.5 (Log10 (FPKM)), and the overall and individual sample FPKM expression distribution is relatively uniform (such as Figure 4 B and C in the figure).

[0045] In the present embodiment, a total of 620 DEGs1 were identified, of which 157 genes were increased in expression in the DN group and 463 genes were decreased in expression. A total of 563 DEGs2 were identified between the DN group and the treatment group. Among them, 339 genes were found to be upregulated and 224 genes were downregulated in the DN group. DEGs1 returned 776 graphene oxide enrichment results, including 619 bp, 69 cellular components (CCs) and 88 molecular functions (MFs). The enrichment results were sorted from small to large by P value, and the top 15 pathways were displayed. In terms of BP, DEGs1 was significantly associated with processes such as "wound healing" and "intermediate filament-based processes"; in terms of CC, DEGs1 was associated with components such as "external encapsulation structure" and "extracellular matrix"; in terms of MF, they were associated with functional pathways such as "oxygen carrier activity" and "fibronectin binding". According to P value < 0.05, DEGs2 obtained 925 GO enrichment results, including 766 bp, 69 CC, and 90 MFs. Again, the top 15 results are shown. Regarding blood pressure, DEGs2 was significantly associated with processes such as “wound healing” and “muscle cell differentiation”; regarding CC, DEGs2 was associated with components such as “extracellular matrix” and “hemoglobin complex”; as for MF, they were associated with functional pathways such as “heparin binding” and “oxygen binding”. In addition, DEGs1 derived 294 KEGG enrichment results, mainly including related pathways such as “motor protein” and “IL-17 signaling pathway”. DEGs2 generated a total of 276 KEGG pathways, such as “cytokine-cytokine receptor interaction” and “ascorbic acid and aldehyde salt metabolism”.

[0046] In the present invention, it was found that LOX, TIMP1, NFKBIA, POSTN and SHH are key targets of DN. Specifically, in the present invention, the intersection of up-regulated DEGs1 and down-regulated DEGs2 produced 172 intersection genes, the intersection of down-regulated DEGs1 and up-regulated DEGs2 produced 39 intersection genes, and the two parts of the intersection genes were juxtaposed to form 211 genes related to the treatment of Chinese medicine composition. Correlation analysis showed that there was a strong interaction between DEGs related to the treatment of Chinese medicine composition. A total of 484 GO enrichment results were obtained for DEGs related to the treatment of Chinese medicine composition, including 407 bp ("oxygen transport", "wound healing", etc.), 32 CC ("extracellular matrix", "collagen trimer", etc.), and 45 MFs ("heparin binding", "oxygen binding", etc.), and the top 15 items performed significantly. At the same time, 226 KEGG enrichment results were obtained, mainly including related pathways such as "basal cell carcinoma" and "small cell lung cancer". In addition, the PPI network has a total of 113 nodes and 139 edges. After screening, the number of genes that meet the five algorithms is 5, and these five key targets are LOX, TIMP1, NFKBIA, POSTN and SHH.

[0047] In the embodiment of the present invention, the key target related genes, TF and miRNA are also predicted. Specifically, there are 20 genes in the GGI network with the strongest functional similarity to 5 key targets (SMO, CEBPD, DHH, GPC3, IHH, TIMP3, etc.), with a total of 243 interactions. It can be seen that the functions involved in the key targets include "collagen-containing extracellular matrix", "extracellular structural organization", "extracellular matrix organization", etc. The TF-key gene network shows the interaction between TF and key targets. It is estimated that there are 30 TFs in the three key targets, and there are 33 nodes and 32 edges in the network. The TF that jointly regulates NFKBIA and SHH is NFKB1, and the TF that jointly regulates SHH and POSTN is TWIST1. In addition, a total of 60 miRNAs of 5 key targets were predicted, and the mRNA-miRNA network had 64 nodes and 70 edges. LOX did not predict miRNA. The miRNA co-regulated by SHH and TIMP1 is rno-miR-193b-5p, the miRNA co-regulated by SHH and POSTN are rno-miR-676, rno-miR-351-5p, and rno-miR-222-5p, the miRNA co-regulated by TIMP1 and POSTN is rno-miR-760-3p, and the miRNA co-regulated by NFKBIA and POSTN is rno-Let-7b-5p.

[0048] In the present embodiment, the expression levels of key targets were detected by RT-qPCR. Specifically, the RT-qPCR results showed that the expression levels of TIMP1, NFKBIA and SHH in the DN group were increased compared with the control group, while the expression levels of these three key target genes in the treatment group were lower than those in the DN group (e.g. Figure 5 As shown, Figure 5 The expression levels of LOX, TIMP1, NFKBIA, POSTN and SHH in three groups of rats are shown in the figure. However, there was no significant difference in the expression levels of LOX and POSTN among the groups.

[0049] Although the mechanism of DN is not fully understood, current evidence has identified several factors, including the accumulation of AGEs, inflammatory damage, oxidative stress, RAAS dysregulation, and fibrosis. These pathological changes create a complex and intertwined network of biological and metabolic interactions that promote the progression of DN. Based on the above exploratory analysis, the present invention proposes that Chinese medicine can enhance DN through multiple targets and pathways, including podocyte protection, anti-inflammation, anti-fibrosis, antioxidant and metabolic regulation. These evidences support the potential advantages of traditional Chinese medicine in treating DN involving multiple pathogenic factors. In the present invention, the potential mechanism of the Chinese medicine composition for the treatment of DN was explored using a combination of transcriptomics and bioinformatics, and it was found that TIMP1, LOX, POSTN, NFKBIA and SHH may be the key targets of the Chinese medicine composition for reducing ECM deposition and alleviating renal fibrosis, providing a reference for the treatment of DN.

[0050] Among them, the tissue inhibitor of metalloproteinases 1 (TIMP1) gene is a member of the TIMP gene family. The protein it encodes acts as a natural inhibitor of matrix metallopeptidases and plays a role in regulating cell apoptosis, migration and differentiation. TIMP1 levels have been considered to be associated with DN in the past. A recent study showed that TIMP1 can be used as a biomarker for oxidative stress and inflammatory response in DN. Excessive accumulation of extracellular matrix (ECM) is considered to be an important pathophysiological factor leading to interstitial fibrosis and glomerular sclerosis, which are typical pathological features of DN. TIMPs and matrix metalloproteinases (MMPs) play a key role in regulating ECM degradation in DN. Among matrix metalloproteinases, matrix metalloproteinase-9 (MMP-9) is one of the main enzymes responsible for degrading type IV collagen. At physiological pH, MMP-9 plays a major role in ECM degradation, while TIMP1 shows high specificity in inhibiting MMP-9. Studies have shown that the activity of MMP9 in glomerular podocytes is reduced under long-term hyperglycemia. A high sugar environment can enhance the non-enzymatic glycosylation of type IV collagen, thereby affecting the expression and activity of MMPs. Glycosylated collagen is insensitive to MMPs and cannot be degraded by them. In addition, there is evidence that a decrease in MMP-9 levels and an increase in TIMP1 expression disrupt the balance between mesangial matrix synthesis and degradation, leading to mesangial expansion. In the examples of the present invention, it was observed that the ECM deposition in renal cells of DN rats treated with a Chinese medicine composition was reduced. RT-PCR results further confirmed that TIMP1 expression was reduced. This suggests that the Chinese medicine composition delays the progression of DN renal fibrosis, which is related to the regulation of TIMP1. One possible explanation is that the reduced expression of TIMP1 inhibits MMP9 and promotes the degradation of ECM.

[0051] Sonic Hedgehog (SHH) is also associated with ECM deposition and fibrosis in renal disease. SHH binds to the patched receptor, thereby relieving the inhibition of smoothened (SMO). Activated SMO then activates Gli transcription factors, promoting their translocation to the nucleus. Within the nucleus, Gli controls gene expression, promotes fibroblast proliferation and differentiation, and increases the production of ECM proteins such as collagen and fibronectin. As mentioned earlier, TGF-β plays a key role in the renal fibrosis pathway. In a high-glucose environment, overexpression of SHH not only promotes ECM synthesis, but also synergizes with the TGF-β signaling pathway to aggravate tubular cell fibrosis. There is a synergistic effect between the SHH and TGF-β pathways. For example, connective tissue growth factor (CTGF) plays a vital role in regulating cell differentiation, proliferation, and ECM protein production, and the activation of Gli transcription factors in the SHH signaling pathway further regulates connective tissue growth factor (CTGF). At the same time, CTGF, as an important downstream target of TGF-β signaling, amplifies the profibrotic effect of TGF-β through a feedback loop. The Chinese herbal composition down-regulates the expression of SHH in DN rat tissues, which may further inhibit the role of the SHH signaling pathway in the progression of renal fibrosis. However, this process requires further studies to quantitatively evaluate the expression of downstream targets in the SHH pathway. Importantly, among the main targets down-regulated by the Chinese herbal composition, POSTN, TIMP1, SHH, and LOX are all associated with the progression of renal fibrosis. Masson staining showed a decrease in collagen fibers, and PAS staining showed a decrease in glomerular mesangial expansion and ECM deposition. These results indicate that the Chinese herbal composition effectively inhibits the progression of DN renal fibrosis through multiple targets.

[0052] NFKBIA is a key regulatory protein that plays an important role in inflammation and immune response by inhibiting the NF-κB signaling pathway. In the case of DN, glomerular and tubulointerstitial inflammation are important pathological features. NF-κB is mainly located in the renal interstitium, glomerulus and tubular epithelial cells. The sustained activation of the NF-κB pathway in DN leads to increased expression of downstream inflammatory mediators, thereby promoting inflammatory response and promoting the progression of DN. In theory, blocking the NF-κB signaling pathway with NFKBIA is helpful for DN. Differential gene analysis showed that NFKBIA expression was upregulated in the DN group compared with the control group, suggesting that there may be a compensatory mechanism in DN to control the progression of renal inflammation. In the examples of the present invention, it was observed that the expression of NFKBIA was downregulated in the treatment group. However, it should be noted that this does not mean that the Chinese medicine composition will aggravate inflammation. In the examples of the present invention, the Chinese medicine composition was observed to have a renal protective effect and a certain anti-fibrosis progression in the rat model. A reasonable explanation is that the Chinese medicine composition may activate other alternative anti-inflammatory or anti-fibrosis signaling pathways, effectively compensating for the regulation of the NF-κB pathway.

[0053] Lysyl oxidase (LOX) is an extracellular enzyme that plays a crucial role in the post-translational modification of collagen and elastin by catalyzing cross-linking reactions and intra-cross-linking reactions. Excessive LOX activity on interstitial collagen leads to cross-linking, which can irreversibly develop into chronic tubulointerstitial fibrosis in mouse kidneys because cross-linked ECM is highly resistant to degrading enzymes. In addition, under hypoxic conditions, activation of HIF-1 signaling upregulates LOX expression, leading to a transition of renal epithelial cells from epithelial to interstitial cells, thereby promoting the development of renal fibrosis. In the case of DN, both glomerular basement membrane thickening and glomerular sclerosis are associated with excessive ECM deposition. In addition to the relationship between Timp1 and ECM discussed above, LOX is also a key target of ECM dysregulation in DN. In the kidneys of rats with early DN, increased LOX expression was observed in both tubules and glomeruli. This elevation of LOX may impair the functional integrity of the glomerular basement membrane while also promoting excessive ECM accumulation. Therefore, LOX is involved in ECM deposition in the early stages of DN as well as the progression of renal fibrosis in the later stages. Periostin (POSTN) is a special extracellular matrix protein that participates in cell-to-cell signal transduction, cell adhesion and migration, and metabolic regulation. High levels of POSTN expression have been found in fibrotic tissues. In the kidney, POSTN is expressed in the tubulointerstitial region, and a study has shown that urinary POSTN levels are associated with tubular damage in renal fibrosis. In addition, under ischemia-reperfusion and hypoxia conditions, POSTN can promote renal fibrosis through the p38MAPK pathway. The link between POSTN and DN has been confirmed in a recent study. Briefly, in the hyperglycemic and inflammatory environment of DN, TGF-β promotes the expression of POSTN, and POSTN further amplifies TGF-β levels, forming a vicious cycle. In the presence of TGF-β, POSTN directly aggravates tubular cell fibrosis and aggravates DN. In addition, it is necessary to mention the classical renal fibrosis pathway, in which TGF-β regulates the expression of fibrosis-related genes and the accumulation of ECM through smad-dependent and smad-independent pathways. Blocking the mutual reinforcement between TGF-β and POSTN is essential to slow the progression of DN fibrosis. RT-PCR results showed no significant differences in the expression of LOX and POSTN between the groups, while transcriptome sequencing of rat kidney tissues treated with the TCM composition showed downregulation of these genes. Transcriptome sequencing generally involves large-scale data analysis that can detect subtle expression changes, while PCR is more precise in gene quantification. Differences in sample size may also lead to nonsignificant RT-PCR results. Nevertheless, LOX and POSTN remain promising targets for further research in the treatment of DN with the TCM composition.

[0054] Based on the above basic research, transcriptome sequencing and bioinformatics methods, the present invention embodiment found evidence that the aforementioned Chinese medicine composition regulates DN through multiple targets. This discovery has important clinical significance for exploring how Chinese medicine can improve DN and seeking the combination of Chinese and Western medicine for the treatment of chronic and complex diseases such as DN.

[0055] Based on the above research, an embodiment of the present invention provides a traditional Chinese medicine composition for treating diabetic nephropathy, wherein the traditional Chinese medicine composition comprises: astragalus, rhubarb, leech, salvia miltiorrhiza and epimedium.

[0056] In the embodiment of the present invention, in practical application, the ratio of the Chinese medicine composition is: astragalus: rhubarb: leech: salvia miltiorrhiza: epimedium = 7:2:1:6:5.

[0057] In the embodiment of the present invention, in actual application, the ratio of various components in the traditional Chinese medicine composition is not limited to the above ratio, and the ratio of various components in the traditional Chinese medicine composition can be adjusted by medical personnel according to actual conditions.

[0058] In the embodiment of the present invention, in actual application, the Chinese medicine composition can be a powder, an aqueous solution, a tablet or a capsule.

[0059] The present invention also provides an application of the above-mentioned Chinese medicine composition in the preparation of a drug for treating diabetic nephropathy. The Chinese medicine composition acts on the targets: TIMP1, LOX, POSTN, NFKBIA and SHH. The Chinese medicine composition is used to reduce ECM deposition and alleviate renal fibrosis to slow down the development of diabetic nephropathy.

[0060] Among them, the miRNA co-regulated by SHH and TIMP1 is rno-miR-193b-5p, the miRNA co-regulated by SHH and POSTN are rno-miR-676, rno-miR-351-5p, and rno-miR-222-5p, the miRNA co-regulated by TIMP1 and POSTN is rno-miR-760-3p, and the miRNA co-regulated by NFKBIA and POSTN is rno-Let-7b-5p.

[0061] Optionally, the Chinese medicine composition inhibits TIMP1 expression, reduces the inhibition of MMP9, and promotes the degradation of ECM.

[0062] Optionally, the Chinese medicine composition inhibits the transformation of renal epithelial cells from epithelial cells to mesenchymal cells by inhibiting the expression of LOX, thereby reducing the occurrence of renal fibrosis.

[0063] Among them, excessive LOX activity on interstitial collagen leads to cross-linking. Since cross-linked ECM is highly resistant to degradative enzymes, cross-linking can irreversibly develop into chronic tubulointerstitial fibrosis in mouse kidneys. Under hypoxic conditions, activation of HIF-1 signaling upregulates LOX expression, causing renal epithelial cells to transform from epithelial cells to interstitial cells, thereby promoting the occurrence of renal fibrosis. The increase in LOX may impair the functional integrity of the glomerular basement membrane and also promote excessive ECM accumulation.

[0064] Optionally, the Chinese medicine composition inhibits the expression of POSTN, slows down renal tubular cell fibrosis, slows down the development of diabetic nephropathy, blocks the mutual reinforcement between TGF-β and POSTN, and slows down the progression of diabetic kidney fibrosis.

[0065] Among them, TGF-β promotes the expression of POSTN, and POSTN further amplifies the level of TGF-β, forming a vicious circle; when TGF-β exists, POSTN directly aggravates tubular cell fibrosis and aggravates DN; TGF-β regulates the expression of fibrosis-related genes and the accumulation of ECM through smad-dependent and smad-independent pathways. By inhibiting the expression of POSTN and blocking the mutual reinforcement between TGF-β and POSTN, the progression of DN fibrosis can be slowed down. Optionally, the Chinese medicine composition achieves kidney protection by inhibiting the expression of NFKBIA, activating other alternative anti-inflammatory signaling pathways, and compensating for the regulation of the NF-κB pathway.

[0066] Optionally, the Chinese medicine composition inhibits SHH expression to reduce ECM synthesis, and the Chinese medicine composition inhibits the role of SHH signaling pathway in the progression of renal fibrosis by inhibiting SHH expression.

[0067] Among them, SHH binds to its receptor and can release the inhibition of smoothened (SMO). The activated SMO then activates the Gli transcription factor and promotes the translocation of the Gli transcription factor to the nucleus. In the nucleus, Gli controls gene expression, promotes fibroblast proliferation and differentiation, and increases the production of ECM proteins such as collagen and fibronectin. The activation of Gli transcription factors in the SHH signaling pathway further regulates connective tissue growth factor (CTGF). At the same time, CTGF, as an important downstream target of TGF-β signaling, amplifies the profibrotic effect of TGF-β through a feedback loop.

[0068] The present invention also provides an application of the above-mentioned Chinese medicine composition in the preparation of a drug for reducing ECM deposition. The Chinese medicine composition inhibits TIMP1 expression, reduces the inhibition of MMP9, and promotes ECM degradation; the Chinese medicine composition inhibits SHH expression to reduce ECM synthesis.

[0069] The present invention also provides an application of the above-mentioned Chinese medicine composition in the preparation of a drug for alleviating renal fibrosis. The Chinese medicine composition inhibits the transformation of renal epithelial cells from epithelial cells to interstitial cells by inhibiting the expression of LOX, thereby alleviating the occurrence of renal fibrosis; the Chinese medicine composition inhibits the expression of POSTN, thereby slowing down renal tubular cell fibrosis and the development of diabetic nephropathy, and blocks the mutual reinforcement between TGF-β and POSTN to slow down the progression of diabetic renal fibrosis; the Chinese medicine composition inhibits the expression of NFKBIA, activates other alternative anti-inflammatory signaling pathways, and compensates for the regulation of the NF-κB pathway to achieve a renal protective effect; the Chinese medicine composition inhibits the expression of SHH, thereby inhibiting the role of the SHH signaling pathway in the progression of renal fibrosis.

[0070] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0071] The above is a detailed introduction to a Chinese medicine composition and application provided by the present invention. Specific examples are used herein to illustrate the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for a person skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation mode and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A Chinese medicine composition comprising: Astragalus, rhubarb, leech, salvia miltiorrhiza and epimedium.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is in the form of powder, liquid, tablet or capsule.

3. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating diabetic nephropathy, characterized in that: The Chinese medicine composition acts on the targets: TIMP1, LOX, POSTN, NFKBIA and SHH, and is used to reduce ECM deposition and alleviate renal fibrosis to slow down the development of diabetic nephropathy.

4. Use of the Chinese medicine composition according to claim 3 in preparing a medicament for treating diabetic nephropathy, characterized in that: The traditional Chinese medicine composition inhibits the expression of TIMP1, reduces the inhibition of MMP9, and promotes the degradation of ECM.

5. Use of the Chinese medicine composition according to claim 3 in preparing a medicament for treating diabetic nephropathy, characterized in that: The Chinese medicine composition inhibits the expression of LOX, thereby inhibiting the transformation of renal epithelial cells from epithelial cells to interstitial cells and reducing the occurrence of renal fibrosis.

6. Use of the Chinese medicine composition according to claim 3 in preparing a medicament for treating diabetic nephropathy, characterized in that: The Chinese medicine composition inhibits the expression of POSTN, slows down the fibrosis of renal tubular cells, slows down the development of diabetic nephropathy, blocks the mutual reinforcement between TGF-β and POSTN, and slows down the progress of diabetic kidney fibrosis.

7. Use of the Chinese medicine composition according to claim 3 in preparing a medicament for treating diabetic nephropathy, characterized in that: The Chinese medicine composition achieves kidney protection by inhibiting the expression of NFKBIA, activating other alternative anti-inflammatory signal pathways, and compensating for the regulation of the NF-κB pathway.

8. Use of the Chinese medicine composition according to claim 3 in preparing a medicament for treating diabetic nephropathy, characterized in that: The Chinese medicine composition inhibits the expression of SHH to reduce the synthesis of ECM. The Chinese medicine composition inhibits the role of the SHH signaling pathway in the progression of renal fibrosis by inhibiting the expression of SHH.

9. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of a drug for reducing ECM deposition, characterized in that: The Chinese medicine composition inhibits the expression of TIMP1, reduces the inhibition of MMP9, and promotes the degradation of ECM; the Chinese medicine composition inhibits the expression of SHH to reduce the synthesis of ECM.

10. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of a drug for alleviating renal fibrosis, characterized in that: The Chinese medicine composition inhibits the transformation of renal epithelial cells from epithelial cells to interstitial cells by inhibiting the expression of LOX, thereby alleviating the occurrence of renal fibrosis; the Chinese medicine composition slows down the fibrosis of renal tubular cells and the development of diabetic nephropathy by inhibiting the expression of POSTN, and blocks the mutual reinforcement between TGF-β and POSTN to slow down the progression of diabetic renal fibrosis; the Chinese medicine composition achieves a renal protective effect by inhibiting the expression of NFKBIA and activating other alternative anti-inflammatory signaling pathways to compensate for the regulation of the NF-κB pathway; the Chinese medicine composition inhibits the role of the SHH signaling pathway in the progression of renal fibrosis by inhibiting the expression of SHH.

Images