Application of haematococcus pluvialis in preparation of medicine for preventing and treating renal fibrosis caused by urinary tract obstruction

Abstract

The invention relates to the technical field of medicines, discloses application of haematococcus pluvialis in preparation of medicines for preventing and treating renal fibrosis caused by urinary tract obstruction, and discloses a method for treating renal fibrosis more safely and effectively. According to the method, the natural anti-oxidation and anti-inflammatory characteristics of haematococcus pluvialis are utilized, so that the side effects of drugs are reduced, and the treatment safety is improved. Meanwhile, the defense mechanism of the body to renal fibrosis is enhanced through the immunoregulation function of the haematococcus pluvialis, and then the treatment effect is improved. The haematococcus pluvialis provided by the invention can be used as a supplement or substitute of an existing treatment method, provides a new treatment scheme for patients, and is especially suitable for patients with poor response to traditional drugs. Besides, the haematococcus pluvialis not only can treat renal fibrosis, but also can prevent the renal fibrosis, so that the progress of chronic kidney diseases is slowed down or prevented to a certain extent, and the living quality of patients is improved.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to the use of Haematococcus pluvialis in preparing a medicine for preventing and treating renal fibrosis caused by urinary tract obstruction. Background Art

[0002] Renal fibrosis is a typical pathological change in chronic kidney disease (CKD), and epithelial-mesenchymal transition (EMT) is the main stage. Tubulointerstitial fibrosis is characterized by tubular atrophy and extracellular matrix (ECM) accumulation in renal tissue. Since the expression level of TGF-β1 (transforming growth factor, β1) in cells is closely related to the production of EMT and ECM, it is considered to be a strong mediator of renal fibrosis and CKD and a potential key driver of renal fibrosis. At the same time, studies have also reported that senescent cells promote the development of fibrotic diseases by secreting senescence-associated phenotypes (SASP). Therefore, inhibiting EMT can synergistically improve the cell cycle and alleviate cell senescence, which is of great significance for further and better treatment of renal injury. Pathological changes in CKD Renal fibrosis is very common. When the ureter is obstructed, urine retention compresses the renal tubules, leading to progressive necrosis of tubular epithelial cells, infiltration of acute and chronic inflammatory cells in the interstitium, and necrotic tubular tissue is gradually replaced by fibrous scars, eventually forming progressive renal fibrosis. Since fibrosis is considered to be irreversible, inhibiting the progression of fibrosis is considered to be a potential strategy to prevent the development of CKD. Currently, renal fibrosis has become a medical problem to be solved urgently, and the present invention proposes a new solution for this. Therefore, finding new drugs for preventing and treating renal fibrosis has important clinical significance. Summary of the invention

[0003] Hoematococcus pluvialis (HP) is a freshwater unicellular green algae rich in natural antioxidants, such as astaxanthin. Recent studies have shown that Haematococcus pluvialis has multiple biological activities such as anti-inflammatory, antioxidant, and immunomodulatory. Based on these characteristics, the use of Haematococcus pluvialis in the preparation of drugs for the prevention and treatment of various diseases has gradually attracted attention. In view of this, the present invention proposes the use of Haematococcus pluvialis in the preparation of drugs for the prevention and treatment of renal fibrosis caused by urinary tract obstruction, aiming to provide a new prevention and treatment strategy to improve the therapeutic effect of renal fibrosis.

[0004] The present invention provides an application of Haematococcus pluvialis or a preparation thereof, wherein the application comprises any one of the following:

[0005] A1) Use of Haematococcus pluvialis in the preparation of a drug for preventing and treating renal fibrosis caused by urinary tract obstruction;

[0006] A3) Application of Haematococcus pluvialis in regulating the expression of SASP factors related to renal fibrosis;

[0007] A2) Use of Haematococcus pluvialis in the preparation of a drug for regulating the expression of SASP factors associated with renal fibrosis;

[0008] A4) Use of Haematococcus pluvialis in a medicament for inhibiting the process of epithelial-mesenchymal transition induced by TGF-β1 in human renal tubular epithelial cells;

[0009] A5) Use of Haematococcus pluvialis to increase the expression of E-cadherin protein in human renal tubular epithelial cells;

[0010] A6) Use of Haematococcus pluvialis in the preparation of a drug for increasing the expression of E-cadherin protein in human renal tubular epithelial cells;

[0011] A7) Use of Haematococcus pluvialis to reduce the expression of Vimentin protein;

[0012] A8) Use of Haematococcus pluvialis in the preparation of a drug for reducing the expression of Vimentin protein;

[0013] A9) Use of Haematococcus pluvialis in the preparation of a medicament for preventing and / or treating diseases associated with renal fibrosis.

[0014] Preferably, the renal fibrosis-related SASP factors are interleukin-6 and interleukin-8.

[0015] Preferably, the disease associated with renal fibrosis is chronic kidney disease or nephrosclerosis caused by urinary tract obstruction.

[0016] The invention also provides a medicine for treating kidney disease, wherein the active ingredient of the medicine is Haematococcus pluvialis.

[0017] Preferably, the renal disease is renal fibrosis, chronic kidney disease or nephrosclerosis caused by urinary tract obstruction.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Provide a safer and more effective method for treating renal fibrosis, by utilizing the natural antioxidant and anti-inflammatory properties of Haematococcus pluvialis, reducing the side effects of drugs and improving the safety of treatment;

[0020] 2. Through the immunomodulatory potential of Haematococcus pluvialis, the body's defense against renal fibrosis is enhanced, thereby improving the therapeutic effect;

[0021] 3. The Haematococcus pluvialis of the present invention can be used as a supplement or substitute for existing treatment methods, providing patients with new treatment options, especially for patients who do not respond well to traditional drugs;

[0022] 4. The preparation process of the Haematococcus pluvialis of the present invention is simple, the cost is low, and it is easy to produce and apply on a large scale, which helps to reduce the cost of treatment and make it affordable for more patients;

[0023] 5. The Haematococcus pluvialis of the present invention can not only treat renal fibrosis, but also prevent its occurrence, thereby slowing down or preventing the progression of chronic kidney disease to a certain extent and improving the quality of life of patients.

[0024] In summary, the present invention provides a new therapeutic strategy for preventing and treating renal fibrosis through the multiple biological activities of Haematococcus pluvialis, and has significant clinical application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0026] Figure 1 HE, Masson, and PAS staining results;

[0027] Figure 2 It is the result of quantitative reverse transcription-polymerase chain reaction;

[0028] Figure 3 The results of immunofluorescence staining. DETAILED DESCRIPTION

[0029] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0030] Example 1 Chemical composition analysis of Haematococcus pluvialis

[0031] In this experiment, the chemical composition of Haematococcus pluvialis (HP, provided by Yunnan Alcon Biotechnology Co., Ltd.) was deeply analyzed by using Thermo QE plus liquid chromatography tandem high-resolution mass spectrometer and Compounddiscover data processing software.

[0032] 1. Sample pretreatment

[0033] 1. Take 200 mg of Haematococcus pluvialis sample and place it in a suitable container.

[0034] 2. Add 10 mL of 50% methanol aqueous solution (methanol to water volume ratio is 50:50) to the container and perform ultrasonic treatment for 30 minutes.

[0035] 3. After the ultrasound, take 1 mL of the supernatant, transfer it to a centrifuge tube, and centrifuge it at 14,000 rpm for 5 minutes.

[0036] 4. After centrifugation, take the supernatant again, filter it through a 0.22 μm microporous filter membrane, and then inject the filtrate into the injection bottle for UHPLC-MS / MS analysis. The blank sample is processed under the same conditions.

[0037] 2. Liquid Phase Conditions

[0038] 1. The chromatographic column used was ACQUITY UPLC HSST3 column (specification: 2.1x100mm, particle size: 1.8μm), and the column temperature was set to 35°C.

[0039] 2. The injection volume was 10 μL and the flow rate was controlled at 0.3 mL / min.

[0040] 3. The mobile phase was divided into phase A (deionized water, containing 0.1% formic acid) and phase B (acetonitrile, containing 0.1% formic acid), and gradient elution was used for separation.

[0041] 3. Mass spectrometry conditions

[0042] When collecting mass spectrometry data, a QExactive Orbitrap high-resolution mass spectrometer was used, and the FullMS-ddMS2 detection mode was set. To ensure the comprehensiveness and accuracy of the data, positive and negative ion modes were scanned, and the scanning range was set to m / z 100-1200.

[0043] In the parameter setting of the mass spectrometer, various parameters were adjusted to ensure the best detection effect. The resolution of MS1 was set to 70,000, and the resolution of MS2 was set to 17,500. This setting ensures that we can obtain high-resolution mass spectrometry data.

[0044] In addition, key parameters such as ion source voltage, capillary ion transfer tube temperature, auxiliary gas heating temperature and gas flow rate were optimized. The ion source voltage was set to 3.2kV ​​to provide a stable ion source. The capillary ion transfer tube temperature (Capillarytemp) was set to 320°C to ensure the stability of ions during transmission. The auxiliary gas heating temperature (Aux gas heater temp) was set to 350°C to further promote the transmission and separation of ions. In terms of gas flow rate, the sheath gas flow rate was set to 40L / min, while the auxiliary gas flow rate (Aux gasflowrate) was set to 15L / min. This setting can ensure the stable flow of gas in the mass spectrometer, thereby improving the stability and sensitivity of detection.

[0045] In terms of triggering conditions for MS2 scanning, a step-type fragmentation voltage NCE was used as the collision energy, and three different energy levels of 30, 40, and 50 were set. This setting can obtain richer fragment ion information, which helps to more accurately analyze and identify the compounds in the sample.

[0046] Finally, AGCTarget was set to 1e6 (i.e., 1 times 10 to the sixth power), and TopN was set to 5. This setting ensures that the top five ions with the strongest signals can be automatically selected for MS2 scanning during mass spectrometry data acquisition, thereby further improving the quality and reliability of the data.

[0047] IV. Experimental Results

[0048] Compound Discover 3.2 software was used to extract characteristic peaks from the original Raw mass spectrometry data, and strict standards were set for the mass deviation of characteristic peak element matching, molecular formula prediction, and isotope distribution matching to be within 5ppm. In order to ensure the accuracy of the identification results, we combined the mzCloud online database with the locally built mzVault Chinese medicine natural product database for characteristic peak identification. The standard for screening positive results was set as a mass deviation of less than 5ppm, consistent with the isotope distribution law, and a matching score of more than 70 points in the mzVault best match database. Table 1 shows the results of the chemical composition analysis of Haematococcus pluvialis. From the data in Table 1, we can clearly observe that the main chemical components in Haematococcus pluvialis are amino acids, among which the content of L-leucine is particularly prominent, with a relative content of up to 47.622%.

[0049] Table 1

[0050]

[0051]

[0052] Example 2

[0053] 1. Experimental Materials

[0054] SD rats were provided by Sibeifu (Beijing) Biotechnology Co., Ltd. (Animal Certificate No.: NO.110324231106182758); Haematococcus pluvialis (HP) was provided by Yunnan Alcon Biotechnology Co., Ltd.

[0055] 2. Experimental Methods

[0056] 42 SPF male SD rats weighing 150±10g were selected and placed in a standard SPF environment. Standard feed was provided and rats were free to drink water. After one week of normal feed adaptation, the animals were randomly divided into six groups, 12 in each group. Seven days before modeling, the UUO+HP group and the UUO+AST group were gavaged with Haematococcus pluvialis (dose of 233.33mg / kg) and astaxanthin (dose of 25mg / kg), respectively, once a day, and fasted for 24 hours after the last gavage. After the fasting, the Con group, UUO group, UUO+HP group, UUO+AST group, and UUO+pirfenidone group were modeled. When the rats were anesthetized, atropine sulfate was subcutaneously injected 15 minutes in advance, followed by an intramuscular injection of Shutai 50 for anesthesia. The rats were fixed on the operating board in a lateral position, and a longitudinal incision was cut along the kidney position to expose the kidney. The normal group was not treated, while the model group and the drug-treated group underwent ureteral ligation. After the operation, the inner and outer skins were quickly sutured and disinfected. After the rats woke up, they were fed routinely and their condition was observed. After the model was successfully established, medication was started. The feeding methods of each group were as follows:

[0057] Control group (sham operation group, also called normal group or Con group): provided with normal water and feed daily;

[0058] UUO group (urinary tract obstruction renal fibrosis model group): normal water and feed were provided daily;

[0059] UUO+HP group (Haematococcus pluvialis group): starting from the second day after surgery, daily gavage of Haematococcus pluvialis was performed at a dose of 233.33 mg / kg, and normal water and feed were provided daily for 14 days;

[0060] UUO+AST group (astaxanthin group): Starting from the second day after surgery, astaxanthin was gavaged daily at a dose of 25 mg / kg, and normal water and feed were provided daily;

[0061] UUO+pirfenidone group (pirfenidone group): Pirfenidone was gavaged daily from the second day after surgery at a dose of 500 mg / kg, and normal water and feed were provided daily.

[0062] During the modeling and the above treatment process, the rats were weighed weekly. On the 14th day of administration, all rats were anesthetized and killed, and the renal cortical tissue was collected. The collected renal cortical tissue was fixed in 4% paraformaldehyde, dehydrated, embedded and sliced ​​to prepare paraffin sections. The sections were stained with hematoxylin-eosin staining (HE), Masson staining and glycogen (Periodic Acid-Schiff stain, PAS) staining.

[0063] HE, PAS and Masson staining were used to evaluate the effects of Haematococcus pluvialis on renal histological changes and mesangial matrix deposition. HE staining showed that compared with normal rats, UUO rats had hydronephrosis, progressive dilatation of the renal pelvis and thinning of the renal cortex. Haematococcus pluvialis, astaxanthin and pirfenidone could improve renal tubular and glomerular damage, and the improvement effect of Haematococcus pluvialis was better than that of astaxanthin and pirfenidone ( Figure 1 A, B). Masson staining showed that collagen deposition in the glomeruli of UUO rats increased ( Figure 1 B), Haematococcus pluvialis, astaxanthin and pirfenidone can reduce glomerular collagen deposition in UUO rats, and the effect of Haematococcus pluvialis is better than that of astaxanthin and pirfenidone ( Figure 1 B). PAS staining showed that the PAS-positive mesangial matrix in the glomeruli of UUO rats increased compared with that in normal rats ( Figure 1 B), Haematococcus pluvialis, astaxanthin and pirfenidone can reduce the PAS-positive mesangial matrix in the glomeruli of UUO rats, and the effect of Haematococcus pluvialis is better than that of astaxanthin ( Figure 1 B).

[0064] Taken together, these results suggest that Haematococcus pluvialis can alleviate renal fibrosis by reducing extracellular matrix (ECM) deposition and improving renal cortical damage, and its effect is better than astaxanthin and pirfenidone.

[0065] Example 3 Haematococcus pluvialis improves the expression of SASP in human renal tubular epithelial cells induced by TGF-β1 at the genetic level

[0066] 1. Experimental Materials

[0067] Includes human renal tubular epithelial cells (provided by Shanghai Meiwan Biotechnology Co., Ltd.) and DMEM / F-12 (1:1) culture medium (purchased from Thermo Fisher Scientific).

[0068] 2. Experimental Methods

[0069] The quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technique was used to determine the expression level of the SASP gene in total RNA extracted from human kidney 2 tubular epithelial cells. The specific operation steps of the quantitative reverse transcription-polymerase chain reaction are conventional techniques in the art, so they will not be repeated here.

[0070] The specific steps of TGF-β1 inducing HK-2 cells are as follows:

[0071] First, HK-2 cells were seeded in a six-well plate with a cell density of approximately 2 × 10 5 The six-well plates were then placed in a cell culture incubator at 37°C with 5% carbon dioxide for 24 hours. Afterwards, the cells were divided into six groups for different treatments:

[0072] 1. Normal control group (Control or Con group): remove the old culture medium and replace it with new culture medium containing 10% fetal bovine serum (FBS), and continue to culture in an incubator at 37°C and 5% carbon dioxide for 48 hours;

[0073] 2. TGF-β1 model group: remove the old culture medium and replace it with 10% FBS culture medium containing 1 nanomole (M) TGF-β1, and culture in an incubator at 37°C and 5% carbon dioxide for 48 hours;

[0074] 3. Low-dose group of Haematococcus pluvialis (TGF-β1+8μg / mL HP group): remove the old culture medium and replace it with 10% FBS medium containing 10 ng / ml TGF-β1 and 8 μg / ml HP. The culture conditions are the same as above for 48 hours.

[0075] 4. Medium-dose group of Haematococcus pluvialis (TGF-β1+16 μg / mL HP group): remove the old culture medium and replace it with 10% FBS culture medium containing 10 ng / ml (ng / ml) TGF-β1 and 16 μg / ml (μg / mL) Haematococcus pluvialis (HP), culture conditions are the same as above, and continue for 48 hours;

[0076] 5. High-dose group of Haematococcus pluvialis (TGF-β1+24 μg / mL HP group): remove the old culture medium and replace it with 10% FBS medium containing 10 ng / ml TGF-β1 and 24 μg / ml HP. The culture conditions are the same as above for 48 hours.

[0077] 6. Astaxanthin group (TGF-β1+16μg / mLAST group): The old culture medium was removed and replaced with 10% FBS culture medium containing 10 ng / ml TGF-β1 and 16 μg / ml astaxanthin (AST). The culture conditions were the same as above and continued for 48 hours.

[0078] After the above-mentioned groups were treated, cell samples were collected and total cellular RNA and total protein were extracted for subsequent experiments.

[0079] 3. Experimental Results

[0080] like Figure 2 As shown in the figure, the trend of quantitative polymerase chain reaction (qPCR) results was basically consistent with the trend of staining results of renal cortical tissue sections in rats with unilateral ureteral obstruction (UUO). In this study, three factors related to the aging-associated secretory phenotype (SASP) were determined at the messenger RNA (mRNA) level: interleukin-6, interleukin-8, and insulin-like growth factor binding protein 3. Compared with the normal control group, the mRNA expression of IL-6 and IL-8 in human renal tubular epithelial cells induced by transforming growth factor-β1 (TGF-β1) was significantly increased ( Figure 2 A, B), while the mRNA expression of IGFBP3 decreased ( Figure 2 C). After HP intervention, the expression of the above SASP factors showed an improvement trend; after AST intervention, the expression was also improved, but at the same dose, the effect of HP was better than AST. Based on the above results, it can be concluded that Haematococcus pluvialis can improve the mRNA expression of SASP factors in human renal tubular epithelial cells induced by TGF-β1, and its effect is better than AST.

[0081] Experimental Example 4 Haematococcus pluvialis improves the expression of E-cadherin and Vimentin proteins in human renal tubular epithelial cells induced by TGF-β1

[0082] 1. Experimental Materials

[0083] Rat renal cortical tissue collected in Experimental Example 2, human renal tubular epithelial cells collected in Experimental Example 3 II. Experimental Methods

[0084] Western Blot (WB) was used to detect the expression of E-cadherin, N-cadherin, and Vimentin proteins in the renal cortical tissues of each group of rats collected in Example 2 and the total protein samples of human renal tubular epithelial cells collected in Example 3. The operation steps of the Western Blot method are conventional techniques in the art and will not be described in detail here.

[0085] At the same time, the human renal tubular epithelial cells collected in Example 3 were tested for the expression of E-cadherin, N-cadherin, and Vimentin proteins by immunofluorescence (IF). The specific steps of cell immunofluorescence detection are as follows: First, the collected cell samples were washed twice with PBS, then fixed with 4% paraformaldehyde for 15 minutes at room temperature, and then permeabilized with 0.1% TritonX-100 in PBS for 10 minutes; then, the cells were blocked with 10% goat serum for 30 minutes, and then incubated with a primary antibody containing 10% goat serum at 4°C overnight; then, washed three times with PBS, and then incubated in a dark box with DyLight 488-coupled AffiniPure goat anti-mouse IgG (H+L) or CY3-coupled AffiniPure goat anti-rabbit IgG (H+L) for 1.5 hours. The nuclei were stained with DAPI staining reagent for 30 minutes. Finally, the coverslips with cells were placed on glass slides coated with anti-fading mounting medium. Images were taken using a fluorescence microscope.

[0086] 3. Experimental Results

[0087] like Figure 3 As shown in the figure, the results of immunofluorescence (IF) staining were basically consistent with the results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) and the staining results of renal cortical tissue sections of rats with unilateral ureteral obstruction (UUO). Vimentin, as an intermediate filament protein, had a higher fluorescence intensity in the control group (Con group), and was significantly expressed in the intercellular contact area, showing a concentrated and specific fluorescence localization. After treatment with transforming growth factor-β1 (TGF-β1), the fluorescence intensity was significantly weakened ( Figure 3 B). After HP intervention, the fluorescence intensity showed a gradient increase with the increase of HP concentration. After AST intervention, the fluorescence intensity was also enhanced, but at the same dose, the effect of HP was better than AST. E-cadherin, as an epithelial cell-cell adhesion protein, had a weak fluorescence intensity in the control group. After TGF-β1 treatment, its fluorescence intensity increased, indicating that cell-cell adhesion increased and the degree of fibrosis was aggravated. After HP intervention, the fluorescence intensity showed a decreasing trend with the increase of HP concentration ( Figure 3 B). After AST intervention, the fluorescence intensity decreased, but at the same dose, HP had a better effect than AST. Based on the above results, Haematococcus pluvialis can improve the epithelial-mesenchymal transition (EMT) process of human renal tubular epithelial cells induced by TGF-β1, and its effect is better than AST.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An application of Haematococcus pluvialis or a preparation thereof, It is characterized in that The application includes any of the following: A1) Use of Haematococcus pluvialis in the preparation of a drug for preventing and treating renal fibrosis caused by urinary tract obstruction; A3) Application of Haematococcus pluvialis in regulating the expression of SASP factors related to renal fibrosis; A2) Use of Haematococcus pluvialis in the preparation of a drug for regulating the expression of SASP factors associated with renal fibrosis; A4) Use of Haematococcus pluvialis in a medicament for inhibiting the process of epithelial-mesenchymal transition induced by TGF-β1 in human renal tubular epithelial cells; A5) Use of Haematococcus pluvialis to increase the expression of E-cadherin protein in human renal tubular epithelial cells; A6) Use of Haematococcus pluvialis in the preparation of a drug for increasing the expression of E-cadherin protein in human renal tubular epithelial cells; A7) Use of Haematococcus pluvialis to reduce the expression of Vimentin protein; A8) Use of Haematococcus pluvialis in the preparation of a drug for reducing the expression of Vimentin protein; A9) Use of Haematococcus pluvialis in the preparation of a medicament for preventing and / or treating diseases associated with renal fibrosis.

2. The use according to claim 1, It is characterized in that The renal fibrosis-related SASP factors are interleukin-6 and interleukin-8.

3. The use according to claim 1, It is characterized in that The disease associated with renal fibrosis is chronic kidney disease or nephrosclerosis caused by urinary tract obstruction.

4. A drug for treating kidney disease, It is characterized in that The active ingredient of the medicine is Haematococcus pluvialis.

5. The drug according to claim 4, It is characterized in that The renal disease is renal fibrosis, chronic kidney disease or nephrosclerosis caused by urinary tract obstruction.

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