Application of Bacteroides acidophilus in the preparation of drugs for the prevention and treatment of renal ischemia-reperfusion injury

Abstract

The present invention discloses acidogenic Bacteroides ( Bacteroides acidifaciens ) in the preparation of drugs for the prevention and treatment of renal ischemia-reperfusion injury. The present invention first discovered that treatment with acidogenic Bacteroides can significantly reduce the levels of creatinine and urea, the number of tubular necrotic cells, and the ratio of granulocyte influx in mice with I / R injury, can significantly downregulate the levels of inflammatory cytokines in mice with I / R injury, inhibit cell apoptosis, alleviate the changes in the levels of oxidative stress factors caused by I / R injury, and can reduce renal fibrosis induced by I / R injury; indicating that acidogenic Bacteroides can be used to prepare drugs for the prevention and treatment of renal ischemia-reperfusion injury, providing a new approach for the prevention and treatment of renal ischemia-reperfusion injury and acute kidney injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of acidogenic Bacteroides in the preparation of drugs for preventing and treating renal ischemia-reperfusion injury. Background Art

[0002] Acute kidney injury (AKI) is a clinically recognized serious disease with a high incidence and increasing mortality in critically ill patients. It is characterized by a rapid decrease in glomerular filtration rate and an increase in serum creatinine. In addition, AKI can cause water and sodium accumulation in the kidneys, multiple electrolyte abnormalities, and renal fibrosis, and eventually develop into chronic kidney disease. In addition, various factors, including oxidative stress, inflammatory processes, and apoptosis, are also involved in the pathophysiology of the development of AKI into chronic kidney disease.

[0003] Ischemia-reperfusion (I / R) injury is tissue damage caused by blood reperfusion. I / R injury of renal tissue is the main predisposing factor for AKI and can occur during renal transplantation, sepsis, and shock. Therefore, effective prevention and treatment of renal I / R injury can help inhibit the occurrence of AKI or relieve AKI symptoms.

[0004] The intestinal microbiota, also known as the gut flora, is composed of a variety of bacteria that have both favorable and unfavorable effects on human health. Recent studies have shown a close connection between kidney health and the gut flora, with previous studies showing that altered gut flora composition can affect the prognosis of glomerular disease and that kidney inflammation is closely associated with increased intestinal permeability and intestinal production of uremic toxins, which can lead to systemic inflammation.

[0005] As is known to all, intestinal flora can be mainly divided into four major categories: Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria; for example, the Chinese invention patent application with publication number CN 11487842 A discloses a Lactobacillus acidophilus KBL409 strain, the culture, lysate or extract of which can prevent or treat kidney disease by reducing renal inflammation, lowering the blood concentration of uremic toxins, reducing proteinuria, restoring renal mitochondrial function and / or inhibiting renal fibrosis; and Lactobacillus acidophilus is a Gram-positive bacterium belonging to the Firmicutes phylum.

[0006] Currently, there is no evidence that Bacteroides acidogenicus ( Bacteroides acidifaciens ) Any reports related to renal I / R injury. Summary of the invention

[0007] The invention aims to provide the use of acidogenic Bacteroides in the preparation of drugs for preventing and treating renal ischemia-reperfusion injury, thereby providing a new approach for the prevention and treatment of renal ischemia-reperfusion injury and acute kidney injury.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0009] Bacteroides acidogenes ( Bacteroides acidifaciens ) in the preparation of drugs for the prevention and treatment of renal ischemia-reperfusion injury.

[0010] The present invention discovers for the first time that treatment with Bacteroides acidophilus can significantly reduce the creatinine and urea levels, the number of tubular necrotic cells, and the proportion of granulocyte influx in I / R injured mice, can significantly downregulate the levels of inflammatory cytokines in I / R injured mice, inhibit cell apoptosis, alleviate the changes in the levels of oxidative stress factors caused by I / R injury, and can reduce renal fibrosis induced by I / R injury; this indicates that Bacteroides acidophilus can be used to prepare drugs for the prevention and treatment of renal ischemia-reperfusion injury, providing a new approach for the prevention and treatment of renal ischemia-reperfusion injury and acute kidney injury.

[0011] Based on the above research findings, the present invention also provides the use of acidogenic Bacteroides in the preparation of drugs for the prevention and treatment of nephritis.

[0012] The present invention also provides the use of acidogenic Bacteroides in preparing drugs for preventing and treating renal fibrosis.

[0013] The present invention also provides the use of acidogenic Bacteroides in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0014] Preferably, the acidogenic Bacteroides is acidogenic Bacteroides DSM 15896.

[0015] Compared with the prior art, the technical effects of the present invention are embodied in:

[0016] The present invention discovers for the first time that treatment with Bacteroides acidophilus can significantly reduce the creatinine and urea levels, the number of tubular necrotic cells, and the proportion of granulocyte influx in I / R injured mice, can significantly downregulate the levels of inflammatory cytokines in I / R injured mice, inhibit cell apoptosis, alleviate the changes in the levels of oxidative stress factors caused by I / R injury, and can reduce renal fibrosis induced by I / R injury; this indicates that Bacteroides acidophilus can be used to prepare drugs for the prevention and treatment of renal ischemia-reperfusion injury, providing a new approach for the prevention and treatment of renal ischemia-reperfusion injury and acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the creatinine level in the plasma of mice after different treatments;

[0018] Among them, Con represents healthy mice, I / R represents renal ischemia-reperfusion injury model mice, I / R+ vehicle represents renal ischemia-reperfusion injury model mice given sterile water by gavage before modeling, I / R+ BA represents renal ischemia-reperfusion injury model mice given Bacteroides acidophilus by gavage before modeling, Crea (μM) represents creatinine (micromoles per liter), *** or ### represents p < 0.001, the same below;

[0019] Figure 2 is the urea level in mouse plasma after different treatments;

[0020] Wherein, Urea (mM) means urea (millimoles per liter);

[0021] Figure 3 The results of PAS staining of mouse kidney tissue sections after different treatments;

[0022] Figure 4 The results of renal tubular necrosis scores in the kidney tissues of mice after different treatments;

[0023] Tubular necrosis score indicates tubular necrosis score, ** indicates p < 0.01, # indicates p < 0.05, the same below;

[0024] Figure 5 The DAB staining results of mouse kidney tissue sections after different treatments;

[0025] Figure 6 The results of the analysis of granulocyte influx ratio in the renal tissue of mice after different treatments;

[0026] Among them, Granulocytes rate (%) indicates the proportion of granulocyte influx (percentage);

[0027] Figure 7 is the expression level of inflammatory cytokine TNF-α in the kidney tissue of mice after different treatments;

[0028] Wherein, TNF-α mRNA expression (fc) indicates the mRNA expression level of TNF-α (expressed as the expression multiple of GAPDH); the same below;

[0029] Figure 8 is the expression level of inflammatory cytokine IL-1β in the renal tissue of mice after different treatments;

[0030] Fig. 9 is the expression level of inflammatory cytokine IL-6 in the renal tissue of mice after different treatments;

[0031] Fig.10 The western blot analysis results of apoptosis-related proteins in the kidney tissue of mice after different treatments;

[0032] Fig.11 is the expression level of apoptosis-related protein C-Caspase3 in the kidney tissue of mice after different treatments;

[0033] Fig.12 is the expression level of apoptosis-related protein Bax in the kidney tissue of mice after different treatments;

[0034] Fig.13 is the expression level of apoptosis-related protein Bcl-2 in the kidney tissue of mice after different treatments;

[0035] Fig.14 is the level of oxidative stress molecule SOD in the kidney tissue of mice after different treatments;

[0036] Fig.15 is the level of oxidative stress molecule GSH in the kidney tissue of mice after different treatments;

[0037] Fig.16 is the level of oxidative stress molecule MDA in the kidney tissue of mice after different treatments;

[0038] Fig.17 The results of Sirius red staining of mouse kidney tissue sections after different treatments;

[0039] Fig.18 The results of western blot analysis of fibrosis-related proteins in mouse kidney tissue after different treatments;

[0040] Fig.19 is the expression level of fibrosis-related protein α-SMA in the renal tissue of mice after different treatments;

[0041] Among them, α-SMA protein expression (fc) refers to the expression level of α-SMA protein (expressed as the expression multiple of GAPDH); the same below;

[0042] Fig. 20 is the expression level of fibrosis-related protein Vimentin in the renal tissue of mice after different treatments;

[0043] Fig.21 The expression level of fibrosis-related protein Collagen I in mouse kidney tissue after different treatments. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0045] Example 1

[0046] 1. Materials and Reagents

[0047] The test materials and reagents used in this example are as follows:

[0048] Experimental subjects: Male C57Bl / 6 wild-type mice, 8-12 weeks old, weighing 25-30 g;

[0049] Bacteroides acidogenes (BA, strain number: DSM 15896): purchased from Ningbo Mingzhou Biotechnology Co., Ltd.;

[0050] Isoflurane, buprenorphine, and heparin were purchased from Sigma-Aldrich, USA;

[0051] Creatinine and urea ELISA kits: purchased from Wuhan Beiyinlai Biotechnology Co., Ltd. and Shanghai Fantai Biotechnology Co., Ltd.;

[0052] Formalin, PAS staining solution, total RNA extraction reagent, and TBST were purchased from Beijing Solebow Technology Co., Ltd.

[0053] DAB staining solution: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0054] Primescript reverse transcription kit and RT-PCR kit: purchased from TaKaRa, Japan;

[0055] SOD, GSH, MDA colorimetric kit, RIPA lysis buffer: purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0056] Sirius red staining solution, Ly6G, Bcl-2, Bax, cleaved (c)-caspase3, Vimentin, α-SMA, Collagen I, GAPDH antibodies, and secondary antibodies were purchased from Abcam, UK.

[0057] 2. Animal medication and modeling

[0058] The mice were divided into four groups: control group, I / R group, I / R+ vehicle group and I / R+ BA group. Bacteroides acidophilus (3*10 8 CFU BA / 200 μL / mouse) was gavaged into each microbiota-depleted I / R+BA group mouse for one week; each microbiota-depleted mouse in the I / R+ vehicle group received an equal volume of sterile water gavage twice a day for one week.

[0059] One week after administration, the mice in the I / R group, I / R+ vehicle group, and I / R+ BA group were anesthetized with inhaled isoflurane, and their renal pedicles were clamped unilaterally or bilaterally for 25 minutes to induce I / R injury. After that, the kidneys were checked to see whether blood flow had been restored. The mice in the control group did not undergo renal pedicle clamping. The mice in each group underwent cardiac puncture, and blood was collected using test tubes containing heparin. Then, the mice were killed by cervical dislocation, and renal tissues were collected for subsequent analysis.

[0060] 3. Plasma biochemical analysis

[0061] The collected blood samples were centrifuged (10000xg, 4°C, 10 minutes) and the supernatant was collected. The creatinine and urea levels in the plasma were then detected using an ELISA kit. The experimental procedures were strictly followed in accordance with the instructions of the kit. The test results are shown in Figure 1 and Figure 2 .

[0062] Depend on Figure 1 and Figure 2 It can be seen that after I / R injury, the plasma creatinine and urea levels of mice in the I / R group and I / R + vehicle group were significantly higher than those in the control group, indicating that renal function was impaired; while compared with the I / R group and I / R + vehicle group, the plasma creatinine and urea levels of the I / R + BA group were significantly decreased, indicating that after BA pre-administration, the degree of increase in plasma creatinine and urea levels in mice due to renal I / R injury was also relatively mild.

[0063] 4. Tissue staining analysis

[0064] The renal tissues of mice in each group were fixed in 10% formalin, dehydrated in 75%-95% ethanol, clarified in xylene, embedded in paraffin and cut into 4 μm thick sections; then, the tissue samples were dewaxed in xylene and treated with 95%-75% ethanol; subsequently, after the tissue samples were digested with amylase and treated with periodic acid for 15 minutes, the sections were stained with PAS staining solution for 30 minutes to evaluate renal tubular necrosis. The observation results of PAS-stained sections are shown in Figure 2. Figure 3 shown.

[0065] The pathologist scored the proportion of necrotic tubules in the cortical and medullary regions in 10 high-power fields in a blinded manner: 0 = 0%, 1 = < 10%, 2 = 10%–25%, 3 = 25%–50%, 4 = 50%–75%, and 5 = > 75%; the scoring results were as follows: Figure 4 shown.

[0066] like Figure 3 and Figure 4 As shown in the data, after I / R injury, the number of tubular necrotic cells in the renal tissue of mice in the I / R group and I / R + vehicle group increased significantly; while compared with the I / R group and I / R + vehicle group, the number of tubular necrotic cells in the renal tissue of mice in the I / R + BA group decreased significantly, indicating that BA pre-administration can inhibit or alleviate tubular cell necrosis caused by I / R injury.

[0067] 5. Immunohistochemical analysis

[0068] Granulocyte influx is a major feature of the inflammatory environment one day after I / R injury and plays an important role in the postischemic kidney.

[0069] To detect the influx of granulocytes in the kidneys of each group of mice, the sections were prepared in the same way as in Part 4. The sections were then digested with 0.25% pepsin and blocked in 5% bovine serum albumin for 1 hour. The sections were incubated with anti-mouse Ly6G antibody at 4°C overnight, and then incubated with secondary antibodies at room temperature for 30 minutes. The sections were then stained with DAB staining solution and observed under an optical microscope (WMS-1033, WUMO, Shanghai, China) at a magnification of 200 times (the results are shown in Figure 2). Figure 5 ), and 10 non-overlapping high-magnification areas were selected to count the number of Ly6G-positive cells (the results are shown in Figure 6 as shown).

[0070] Depend on Figure 5 and Figure 6 As shown, compared with those in the control group, the number of Ly6G-positive cells in the kidneys of mice in the I / R group and I / R + vehicle group was significantly increased, indicating that I / R injury promoted the influx of granulocytes; while compared with those in the I / R group and I / R + vehicle group, the number of Ly6G-positive cells in the kidneys of mice in the I / R + BA group was significantly decreased, indicating that BA pre-administration could inhibit or alleviate the influx of granulocytes caused by I / R injury.

[0071] 6. Analysis of inflammatory cytokine expression levels

[0072] qRT-PCR was used to detect the expression levels of inflammatory cytokines in the renal tissue of each group of mice to analyze the effect of BA on renal tissue inflammation.

[0073] The analysis method was as follows: total RNA was isolated from renal tissue using an RNA extraction kit, and RNA was quantified using a UV spectrophotometer (DR6000, HACH, Shanghai, China). Then, complementary DNA (cDNA) was synthesized using a Primescript™ reverse transcription kit. qRT-PCR was then performed using SYBR Premix Ex Taq II in a StepOne Plus Real-Time PCR System (4376600, Thermo Fisher, Waltham, Massachusetts, USA). The primer information used for qRT-PCR is shown in Table 1.

[0074] Table 1 qRT-PCR primer information

[0075]

[0076] Pass 2 -ΔΔctThe messenger RNA (mRNA) expression levels of inflammatory cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were detected by the method and normalized to the expression multiple of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The detection results are shown in Figure 7 , Figure 8 and Fig. 9 .

[0077] Depend on Figures 7 to 9 It can be seen that compared with the control group, the expression levels of TNF-α, IL-1β and IL-6 in the renal tissue of mice in the I / R group and I / R + vehicle group were significantly upregulated; compared with the I / R group and I / R + vehicle group, the expression levels of TNF-α, IL-1β and IL-6 in the renal tissue of mice in the I / R + BA group were significantly downregulated, indicating that BA pre-administration can effectively alleviate the inflammatory response caused by I / R injury.

[0078] 7. Analysis of apoptosis-related protein levels

[0079] In this study, Western blot was used to detect the levels of apoptosis-related proteins in the renal tissues of each group of mice to analyze the effects of BA on cell apoptosis after renal I / R injury.

[0080] The analysis method was as follows: total protein was extracted from cells using RIPA lysis buffer, followed by quantification, separation, membrane transfer, and blocking steps; the membrane was then incubated with primary antibodies (Bcl-2, Bax, c-caspase3, GAPDH) at 4°C overnight; the membrane was washed with TBST and incubated with secondary antibodies for 1 hour; protein color was developed using a chemiluminescent substrate (#34577, Thermo Fisher, Waltham, USA), and the results were analyzed on an Odyssey M imaging system (LI-CORBiosciences, Lincoln, Nebraska, USA). The color development results are shown in Fig.10 The analysis results are shown in Fig.11 , Fig.12 and Fig.13 .

[0081] Depend on Figures 10 to 13It can be seen that compared with the control group, the expression levels of Bax and c-caspase3 in the renal tissue of mice in the I / R group and I / R+ vehicle group were significantly upregulated, while the expression level of Bcl-2 was significantly downregulated; compared with the I / R group and I / R+vehicle group, the expression levels of Bax and c-caspase3 in the renal tissue of mice in the I / R+ BA group were significantly downregulated, while the expression level of Bcl-2 was significantly upregulated; this showed that BA could inhibit the expression of Bax and c-caspase3 and promote the expression of Bcl-2, that is, BA pre-administration could effectively inhibit cell apoptosis caused by I / R injury.

[0082] 8. Analysis of oxidative stress levels

[0083] The level of oxidative stress was evaluated by detecting the levels of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in renal tissue to analyze the effect of BA on oxidative stress after renal I / R injury.

[0084] In this example, a colorimetric kit was used to detect the levels of SOD, GSH and MDA in renal tissue: tissue samples were homogenized with PBS and centrifuged at 4°C, and the supernatant was collected and the levels of various oxidative stress factors were detected according to the instructions of the colorimetric kit. Fig.14 , Fig.15 and Fig.16 .

[0085] Depend on Figures 14 to 16 It can be seen that compared with the control group, the SOD and GSH levels in the renal tissue of mice in the I / R group and I / R + vehicle group were significantly downregulated, and the MDA level was significantly upregulated; compared with the I / R group and I / R + vehicle group, the SOD and GSH levels in the renal tissue of mice in the I / R + BA group were significantly upregulated, and the MDA level was significantly downregulated, indicating that BA pre-administration can alleviate the changes in the levels of oxidative stress factors caused by I / R injury.

[0086] 9. Renal fibrosis analysis

[0087] In this example, the Sirius red staining method was used to analyze the renal fibrosis of each group of mice to understand the effect of BA on renal fibrosis after I / R injury.

[0088] The analysis method was as follows: paraffin sections of renal tissue were dewaxed and rehydrated, and then stained with Sirius red stain for 1 hour; the sections were then dehydrated, clarified in xylene, and sealed in neutral gel; the stained areas were observed under a confocal microscope (AX / AX, Nikon, Tokyo, Japan) at a magnification of 200 times, and the observation results were as follows Fig.17 shown.

[0089] Depend on Fig.17 It can be seen that compared with the control group, the number of renal cells stained with Sirius red in the renal tissue of mice in the I / R group and I / R+ vehicle group was significantly increased, indicating that there was obvious renal fibrosis in the I / R mouse model; and compared with the I / R group and I / R+ vehicle group, the number of renal cells stained with Sirius red in the renal tissue of mice in the I / R+ BA group was significantly reduced, indicating that BA pre-administration can alleviate renal fibrosis induced by I / R injury.

[0090] 10. Analysis of fibrosis-related protein levels

[0091] To further verify how BA affects renal fibrosis in I / R mice, Western blotting was used to detect the expression levels of fibrosis-related proteins (α-SMA, Vimentin, and Collagen I) in mouse renal tissue (GAPDH was used as an internal reference). The test results are shown in Fig.18 , Fig.19 , Fig. 20 and Fig.21 .

[0092] like Figures 18 to 21 As shown in the data, compared with those in the control group, the expression levels of α-SMA, Vimentin and Collagen I proteins in the renal tissue of mice in the I / R group and I / R + vehicle group were significantly upregulated; whereas compared with those in the I / R group and I / R + vehicle group, the expression levels of α-SMA, Vimentin and Collagen I proteins in the renal tissue of mice in the I / R + BA group were significantly downregulated, indicating that BA pre-administration alleviated I / R injury-induced renal fibrosis by inhibiting the expression of α-SMA, Vimentin and Collagen I.

Claims

1. Bacteroides acidogenicus ( Bacteroides acidifaciens ) in the preparation of a drug for preventing and treating renal ischemia-reperfusion injury, characterized in that: The acidogenic Bacteroides is acidogenic Bacteroides DSM 15896.

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