Application of herba epimedii extract in reducing toxic and side effects of tripterygium wilfordii

Abstract

The invention discloses application of herba epimedii extract to reduction of toxic and side effects of tripterygium wilfordii. Tests find that the herba epimedii extract can remarkably relieve testis structure damage induced by tripterygium wilfordii polyglycoside TG; the epimedium can reduce the down-regulation of TG-induced reproduction related genes, and the epimedium can increase the expression of genes such as Tssk3, Tex43, sperm1, Atp1a4 and Stata6. In addition, the epimedium extract can increase gene expression of glucose-6-phosphate isomerase (Gpi1), glycolysis provides energy for normal physiological activities of the testis, and epimedium may restore glycolysis and energy supply of testis cells by up-regulating expression of Gpi1. And the epimedium extract can obviously up-regulate the mRNA expression of the complement regulatory protein CD59b. The invention provides a scientific basis for the clinical combined application of the tripterygium glycosides and the herba epimedii extract and the reduction of the toxic and side effects of the reproduction of tripterygium wilfordii.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to application of Epimedium extract in reducing side effects of Tripterygium. BACKGROUND

[0002] Tripterygium is a commonly used traditional Chinese medicine for treating immunological and inflammatory diseases. Tripterygium glycosides (TG) is a total glycoside extracted from Tripterygium. Tripterygium glycosides tablets are a modern traditional Chinese medicine preparation of Tripterygium, which is widely used in the treatment of rheumatoid arthritis, nephrotic syndrome, diabetic nephropathy and the like. Due to the pungent and cold nature of Tripterygium, it is reported to cause liver, kidney and reproductive system toxicity, which limits its wide application.

[0003] In recent years, it has been found that Tripterygium glycosides induces reproductive toxicity through a complex mechanism. The mechanism of reproductive damage induced by Tripterygium glycosides involves multiple signaling pathways such as inflammatory response, oxidative stress, apoptosis, ferroptosis and metabolic imbalance. Glycolysis is the basis of cellular energy metabolism and signal transduction. Under various physiological and pathological conditions, glycolysis is regulated to adapt to different energy requirements and metabolic conditions. Some substances may exhibit toxicity or toxicity attenuation effect by regulating glycolysis. The complement system is an important part of the human immune system, which is activated by three pathways, namely the classical pathway, the alternative pathway and the lectin pathway. All these complement pathways ultimately lead to the formation of membrane attack complex (MAC). MAC is the end product of complement signaling pathway activation, which is composed of C5b, C6, C7, C8 and C9 molecules. MAC insertion into the cell membrane leads to inflammation and cell death. This process is particularly important for the body to fight pathogenic infection. However, the formation of MAC can also cause tissue damage and autoimmune diseases.

[0004] Studies have shown that Tripterygium glycosides can induce testicular cell autophagy and apoptosis. However, it is still unclear whether glycolysis and complement pathways are involved in the reproductive toxicity of Tripterygium glycosides and whether Epimedium can play a detoxification role by regulating glycolysis and complement. SUMMARY

[0005] OBJECTIVE The present application evaluates the protective effect of Epimedium on testicular damage induced by Tripterygium glycosides TG in mice and further explores the potential mechanism. The results show that Epimedium can alleviate TG-induced testicular toxicity by regulating reproductive genes, glycolysis and complement pathways. Epimedium can increase the expression of complement regulatory protein CD59b and glycolysis-related gene Gpi1, thereby inhibiting TG-induced testicular damage.

[0006] The present application is screened through a large number of experiments, and the results show that the Epimedium extract can significantly improve the decrease of testis index and epididymis index induced by Tripterygium glycosides, and reduce the pathological damage of testis induced by Tripterygium glycosides. Fluorescence quantitative PCR analysis shows that the Epimedium extract can induce significant up-regulation of reproductive genes, thereby reducing the testicular toxicity induced by Tripterygium glycosides.

[0007] Technical scheme: In order to achieve the above object, the technical scheme adopted by the present application is: A pharmaceutical composition is prepared from Epimedium extract and Tripterygium glycosides.

[0008] The mass ratio of the Epimedium extract and the Tripterygium glycosides in the above-mentioned pharmaceutical composition is 2:1 to 4:1.

[0009] Application of Epimedium in reducing side effects of Tripterygium.

[0010] As a preferred scheme, the application of Epimedium extract in reducing side effects of Tripterygium glycosides.

[0011] As a particularly preferred scheme, the application of Epimedium extract in reducing reproductive side effects of Tripterygium glycosides.

[0012] The Tripterygium glycosides described in the present application include diterpenoids, triterpenoids and alkaloids of Tripterygium. Preferably, it is a clinically marketed Tripterygium glycoside tablet.

[0013] The Epimedium extract described in the present application is prepared by the following method: taking Epimedium medicinal materials, crushing and sieving, adding 5 to 12 times the volume of water for heating reflux extraction for 1 to 3 times, each time for 1 to 2 hours, filtering, combining the filtrates, reducing pressure concentration, freeze-drying the concentrated solution, and sealing for storage.

[0014] As a more preferred scheme, the Epimedium extract is prepared by the following method: taking Epimedium medicinal materials, crushing and sieving through a No. 3 sieve, adding 10 times the volume of water for heating reflux extraction for 1.5 hours, filtering, continuing to add 10 times the volume of water to the residue for heating reflux extraction for 1.5 hours, filtering. Combining the two filtrates, reducing pressure concentration, freeze-drying the concentrated solution, and sealing for storage.

[0015] Beneficial effects: The present application has the following technical advantages: The present application finds that the Epimedium extract can significantly reduce the testicular structure damage induced by Tripterygium glycoside TG; The Epimedium reduces the down-regulation of reproductive related genes induced by TG, and the Epimedium can increase the gene expression of Tssk3, Tex43, sperm1, Atp1a4 and Spata6. These genes are all involved in the reproductive process such as spermatogenesis. Therefore, the Epimedium can reduce the reproductive toxicity induced by TG by up-regulating the expression of reproductive related genes.

[0016] Epimedium extract can increase the expression of glucose-6-phosphate isomerase (Gpi1) gene, glycolysis provides energy for the normal physiological activity of testis, and Epimedium may restore the glycolysis and energy supply of testicular cells by up-regulating the expression of Gpi1. And Epimedium extract can significantly up-regulate the mRNA expression of complement regulatory protein CD59b.

[0017] In summary, Epimedium can alleviate the reproductive toxicity of TG by regulating reproductive genes, glycolysis and complement. The present application provides a scientific basis for the combined use of Tripterygium glycosides and Epimedium extract in clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 TG induced significant testicular damage. (A) Animal experiment process. (B) Body weight changes of rats in each group. (C) Testicular index. (D) Epididymal index. (E) Representative images of testes in the control group, model group and Epimedium treatment group. Data are expressed as mean SD (n = 6). Statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group; # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 compared with the model.

[0019] Figure 2 Pathohistological staining images of testes in the control group, model group and Epimedium treatment group (scale bar = 50 microns).

[0020] Figure 3 RNA-Seq and GO analysis of differential genes. (A) Volcano plot of deg in the control and model. (B) GO enrichment analysis of DEGs in the control and model. (C) Volcano plot of deg in Epimedium and model. (D) GO enrichment analysis of differential genes in Epimedium and model.

[0021] Figure 4 Epimedium alleviated the decrease in reproductive genes induced by TG. The relative mRNA levels of spermatogenesis (A) and reproductive process (B) genes are shown in the heat map. (C) RT-qPCR analysis of the relative mRNA levels of Tssk3, Tex43, Sperm1, Atp1a4, Spata6 and Tnp1. Statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group; # P<0.05, ## P<0.01 compared with the model; ns, not significant.

[0022] Figure 5 Epimedium supplemented the disordered level of glycolysis. (A) KEGG enrichment analysis of DEGs in the control and model. (B) KEGG enrichment analysis of DEGs in Epimedium and model. (C) Relative mRNA levels of glycolysis genes are shown as a heatmap. (D) RT-qPCR analysis of relative mRNA levels of glycolysis-related genes. Statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group; # P<0.05, ## P<0.01 compared with the model; ns, meaningless.

[0023] Figure 6 Epimedium upregulates the levels of complement regulatory proteins. (A) The relative mRNA levels of complement genes analyzed by RNA-Seq are shown as a heatmap. (B) The relative mRNA level of the complement-related gene CD59b. Statistical significance: *P<0.05, **P<0.01, ***P<0.001 compared with the control group; # P<0.05, ## P<0.01, ### P<0.001 compared with the model. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims. Example 1

[0025] 1. Materials and Methods 1.1 Animal administration Eight-week-old male Balb / c mice were acclimatized for one week and then randomly divided into three groups using a random number table: a control group (0.9% sodium chloride), a Tripterygium wilfordii glycoside model group (TG, 40.5 mg / kg), and a Tripterygium wilfordii glycoside combined with Epimedium extract group (TG 40.5 mg / kg + Epimedium extract 150 mg / kg). The intervention method was gavage for 3 weeks.

[0026] 2.2. Materials and Reagents Tripterygium wilfordii polyglycoside tablets, Zhejiang Deende Pharmaceutical Co., Ltd., National Drug Approval Number Z33020422.

[0027] How is the extract of Epimedium obtained: Take Epimedium medicinal materials, crush through No. 3 sieve, add 10 times the volume of water and heat reflux extract for 1.5 h, filter, continue to add 10 times the volume of water to the residue and heat reflux extract for 1.5 h, filter. Combine the two filtrates, concentrate under reduced pressure, freeze-dry the concentrated solution, and seal for storage.

[0028] 2.2. Body weight change and testis index The body weight of mice was measured every 3 days to obtain the body weight change curve. After the end of drug intervention, the final body weight of the animals was recorded. Then, the animals were euthanized after isoflurane anesthesia. The testis and epididymis weights were recorded, and the testis and epididymis indices were calculated.

[0029] 2.4. Hematoxylin-eosin (H&E) staining After the end of gavage, the mice were sacrificed, the testes were taken and fixed in 4% formaldehyde for 48 h, and stained using hematoxylin and eosin (H&E) staining with a standard protocol. The morphological structure of the testes was observed under a microscope.

[0030] 2.5. RNA extraction The mRNA of the testis tissue of rats in each group was extracted with Trizol reagent, and the concentration and purity of the extracted RNA were detected with Nanodrop 2000. The extracted mRNA was stored at -80°C for RNA sequencing and RT-qPCR verification.

[0031] 2.6. RNA-Seq analysis Three testis mRNA samples were randomly selected from each group for RNA-Seq analysis. After the total RNA sample was qualified, magnetic beads with Oligo(dT) were selected for enrichment and purification. Fragmentation buffer was added to the purified mRNA to randomly break the mRNA into small fragments around 300 bp. Then, the first and second strands were reverse transcribed using the mRNA fragments as templates. Under the action of reverse transcriptase, the mRNA template synthesized single-stranded cDNA through random primers, and then formed stable double-stranded structures through double-strand synthesis. After end repair, base A and sequencing adapters were added, and the products after adapter ligation were purified and fragment sorted. Then, the sorted products were subjected to PCR amplification to obtain the final library. The library was quantified, and RNA-Seq analysis was performed using the NovaSeq X Plus platform. DESeq2 was applied for gene differential expression analysis.

[0032] 2.7. RT-qPCR HiScript III RT SuperMix kit was used for reverse transcription (RT) reaction. RNase-free water and mRNA template sample (100 ng) and 4 μL 4×gDNA Wipeout Mix were added to a 0.2 mL sterile and RNase-free tube. The reaction mixture was mixed gently, and genomic DNA was removed at 42℃ for 2 min. Then 4 μL 5×hiscriptⅲqRT super mix was added to the tube, and after gentle mixing, it was placed in a PCR instrument for RT, with the program set to 37℃ for 15 min, then 85℃ for 5 s. The obtained cDNA product can be used immediately for subsequent qPCR reaction.

[0033] A 20 μL qPCR reaction system was prepared using the following components: 10 μL 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL forward primer (10 microns) and reverse primer (10 microns), 1 μL cDNA, 8.2 μL RNase-free water. The reaction solution was added to a 0.2 mL PCR tube, and PCR amplification was performed in two steps: after denaturation of the product (95℃ for 30 s, 40 cycles per cycle, 95℃ for 5 s, 60℃ for 20 s) amplification; the melting stage was raised to 95℃ for 5 s, the cooling rate was reduced to 65℃ for 1 min at 2.2℃ / s, and the reaction rate was increased to 97℃ for 15 s at 0.11℃ / s.

[0034] Table 1 RT-qPCR primer sequences

[0035] 2.8. Statistical analysis SPSS 26.0 software was used for statistical analysis. All experiments were repeated at least three times, and the results were expressed as mean ± SD. One-way ANOVA was used to show the differences between groups (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0036] 3. Results 3.1. Epimedium can reduce the reproductive toxicity induced by TG The whole experimental process is shown in Figure 1 A. During the entire experimental period, the body weight of mice in each group showed a downward trend Figure 1 B). As shown in Figure 1 C, D, the testis and epididymis indexes of the TG model group were decreased, while the testis and epididymis indexes of the TG + Epimedium group (EbM group) were increased. Histological observation showed that TG induced obvious damage to the testis, and Epimedium intervention could reduce the testicular damage induced by TG Figure 2). The testis structure of the control group was intact, and the number of spermatogenic cells and interstitial cells was normal. The testis structure of the TG treatment group was significantly damaged, and TG-induced testicular toxicity was obvious.

[0037] The above description shows that the combination therapy with Epimedium can significantly reduce the damage of TG-induced testis structure. Therefore, the combination of Epimedium can reduce the reproductive toxicity induced by TG.

[0038] 3.2. RNA-Seq and GO analysis of differential genes In order to clarify the potential detoxification mechanism of Epimedium, the present application performed RNA-seq analysis on the testis of mice. Compared with the control group, 6121 genes were up-regulated and 5552 genes were down-regulated in the TG treatment group Figure 3 A) Compared with the TG model group, 2753 genes were increased and 252 genes were decreased in the Epimedium treatment group Figure 3 C).

[0039] GO enrichment analysis between the TG model group and the control group showed that the obtained differential genes were related to immune cell activation and nucleotide biosynthesis Figure 3 B). Compared with the model group, the differential genes obtained in the Epimedium group were mainly concentrated in the processes related to sperm production, sperm motility and reproductive processes Figure 3 D).

[0040] 3.3. Epimedium reduces the down-regulation of TG-induced reproductive-related genes As shown in Figure 4 A and B, the differential gene heat map analysis showed that the genes related to sperm production and reproductive processes were significantly down-regulated in the TG model group, and were significantly up-regulated in the Epimedium intervention group. These changes were verified by RT-qPCR analysis. As shown in Figure 4 C, it is confirmed that Epimedium can increase the gene expression of Tssk3, Tex43, sperm1, Atp1a4 and Spata6. These genes are all involved in reproductive processes such as spermatogenesis. Therefore, Epimedium can reduce the reproductive toxicity induced by TG by up-regulating the expression of reproductive-related genes.

[0041] 3.4. Epimedium restores the glycolysis disorder induced by TG KEGG enrichment analysis showed that glycolysis, complement and coagulation cascade, immune activation, apoptosis, MAPK and PI3K-Akt were all related to the damage of mouse testis induced by TG Figure 5 A and B). The deg was drawn as a heat map by the present application. As shown in Figure 5C. The TG model group showed significant downregulation of glycolysis-related genes, while the Epimedium group showed significant upregulation. RT-qPCR was used to verify the expression of differential genes. The results showed that the TG group had reduced expression of glucose kinase (Gck), hexokinase 1 (Hk1), phosphoglycerate kinase 2 (Pgk2), and glucose-6-phosphate isomerase (Gpi1) genes Figure 5 D). After treatment with Epimedium, the Gpi1 gene was significantly increased. Glycolysis provides energy for the normal physiological activities of the testis. Therefore, Epimedium may restore glycolysis and energy supply in testicular cells by upregulating the expression of Gpi1.

[0042] 3.5. Epimedium increased the mRNA expression of complement regulatory protein CD59b In addition to regulating glycolysis, the present application also found that Epimedium can regulate complement activation in the testis. As shown in Figure 6 A, complement regulatory proteins (CD46, CD55, CD59) that can inhibit complement activation showed a downward trend in the model group and an upward trend in the Epimedium group. The TG treatment group showed upregulation of C1q, C2, C3, C4, and C7 expression. RT-qPCR analysis was used to verify these changes in complement-related genes. Notably, Epimedium significantly upregulated the mRNA expression of complement regulatory protein CD59b Figure 6 B). These results demonstrate that TG induces complement activation, and Epimedium upregulates the expression of complement regulatory protein CD59b.

[0043] The above studies show that the combined use of Epimedium extract can reduce the toxicity of TG on the testis. The mechanism was verified by RNA-seq and RT-qPCR, and the results suggest that Epimedium can alleviate the reproductive toxicity of TG by regulating reproductive genes, glycolysis, and complement. The present application provides a scientific basis for the combined use of Tripterygium and Epimedium in clinical practice and a reference for reducing the reproductive toxicity of Tripterygium multiglycoside through compatibility in clinical practice.

Claims

1. A pharmaceutical composition, characterized in that, It is made from epimedium extract and tripterygium glycosides.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of Epimedium extract to Tripterygium wilfordii polyglycosides is 2:1 to 4:

1.

3. Application of Epimedium in reducing the toxic side effects of Tripterygium wilfordii.

4. Application of Epimedium extract in reducing the toxic side effects of Tripterygium wilfordii polyglycosides.

5. Application of Epimedium extract in reducing the reproductive toxicity of Tripterygium wilfordii polyglycosides.

6. The application according to any one of claims 3 to 5, characterized in that, Epimedium extract reduces the reproductive toxicity of Tripterygium wilfordii polyglycosides by increasing the gene expression of Tssk3, Tex43, sperm1, Atp1a4, and Spata6.

7. The application according to any one of claims 3 to 5, characterized in that, Epimedium extract reduces the reproductive toxicity of Tripterygium wilfordii polyglycosides by increasing the expression of glucose-6-phosphate isomerase Gpi1 gene.

8. The application according to any one of claims 3 to 5, characterized in that, Epimedium extract reduces the reproductive toxicity of Tripterygium wilfordii polyglycosides by upregulating the mRNA expression of complement regulatory protein CD59b.

9. The application according to any one of claims 3 to 5, characterized in that, The Epimedium extract is prepared by the following method: take Epimedium medicinal material, crush and sieve it, add 5 to 12 times the volume of water, heat and reflux to extract 1 to 3 times, each time for 1 to 2 hours, filter, combine the filtrates, concentrate under reduced pressure, freeze dry the concentrate, and seal and store for later use.

10. The application according to any one of claims 3 to 5, characterized in that, The aforementioned Tripterygium wilfordii polyglycosides include Tripterygium wilfordii diterpenoids, triterpenoids, and alkaloids.

Images