Apoptosis vesicles play role in treating diseases by generating hydrogen sulfide

Abstract

The invention belongs to the field of biological medicines, and relates to an apoptosis vesicle for treating diseases by generating hydrogen sulfide. The invention provides an application of induced vesicles in preparation of antioxidant products or an effect of the induced vesicles in treatment of chronic kidney diseases. The research finds that the induced vesicles generate key enzymes of H2S and generate a significant amount of H2S, and the effect of regulating body functions or treating diseases is achieved by generating H2S.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to an apoptotic vesicle that plays a role in treating diseases by producing hydrogen sulfide. Background Art

[0002] In adults, more than 50-70 billion cells undergo apoptosis every day. Apoptosis is one of the modes of programmed cell death (PCD) that contributes to the regular cell renewal in organisms to maintain tissue repair and immune homeostasis, and plays an important role in development, physiological pathogenesis, and aging. As apoptosis proceeds, cells produce a large number of double-membrane-coated cell structures, previously known as apoptotic bodies (apoBDs); in addition, there is recent evidence that vesicle products called apoptotic vesicles (ApoVs) are also produced during the decomposition of apoptotic cells. Based on its unique production mechanism, ApoVs are able to encapsulate various functional components of its parent cells, including proteins and lipids, which contribute to intercellular communication and maintenance of systemic homeostasis. With the continuous confirmation of the unique beneficial properties of ApoVs, a series of preclinical studies have shown that ApoVs, especially those derived from mesenchymal stem cells (MSCs), have great therapeutic potential in wound repair, tissue regeneration, and immune regulation. However, it is not known whether apoptotic cells and their metabolites ApoVs have antioxidant effects. Summary of the invention

[0003] In some embodiments, the present invention provides the use of inducible vesicles in the preparation of antioxidant products.

[0004] In some embodiments, the vesicles are vesicles produced when stem cells are in normal survival and apoptosis is induced by external factors.

[0005] In some embodiments, the induction method comprises adding staurosporine, ultraviolet irradiation, starvation, or heat stress.

[0006] In some embodiments, the stem cells are mesenchymal stem cells.

[0007] In some embodiments, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

[0008] In some embodiments, the product includes medicine, food, health care product, skin care product, or medical and aesthetic product.

[0009] In some embodiments, the anti-oxidation comprises reducing the MDA content, or reducing the ROS activity, or increasing the SOD activity, or increasing the glutathione peroxidase activity.

[0010] In some embodiments, the antioxidant comprises antioxidant for cells.

[0011] In some embodiments, the cells include skin fibroblasts, stem cells, or vascular endothelial cells.

[0012] In some embodiments, the stem cells comprise mesenchymal stem cells.

[0013] In some embodiments, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

[0014] In some embodiments, the diameter of the vesicle is 0.03-10 μm. In some embodiments, the diameter of the vesicle is 0.03-6 μm. In some embodiments, the diameter of the vesicle is 0.03-4.5 μm. In some embodiments, the diameter of the vesicle is 0.03-1 μm. In some embodiments, the concentration of staurosporine is 1-15000 nM. In some embodiments, the concentration of staurosporine is 200-10000 nM. In some embodiments, the concentration of staurosporine is 500-1000 nM. In some embodiments, the concentration of staurosporine is 500-900 nM. In some embodiments, the concentration of staurosporine is 500-800 nM. In some embodiments, apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 1 to 900 nM. In some embodiments, apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 10 to 800 nM. In some embodiments, apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 100 to 800 nM. In some embodiments, apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 200 to 700 nM. In some embodiments, apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 300 to 600 nM. In some embodiments, apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 400 to 600 nM.

[0015] In some embodiments, the vesicles express a marker Syntaxin 4 at a higher level than MSCs or exosomes of the same origin. In some embodiments, the markers further include one or more of Annexin V, Flotillin-1, Cadherin 11, or Integrin alpha 5. In some embodiments, the vesicles highly express markers Annexin V, Flotillin-1, Cadherin 11, or Integrin alpha 5. In some embodiments, the induced vesicles express markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 at a higher level than MSCs or exosomes of the same origin.

[0016] In some embodiments, the present invention provides the use of inducible vesicles in the preparation of a drug for treating chronic kidney disease.

[0017] In some embodiments, the chronic kidney disease is chronic nephritis.

[0018] In some embodiments, the chronic kidney disease is glomerulonephritis.

[0019] In some embodiments, the vesicles are vesicles produced when stem cells are in normal survival and apoptosis is induced by external factors.

[0020] In some embodiments, the induction method comprises adding staurosporine, ultraviolet irradiation, starvation, or heat stress.

[0021] In some embodiments, the stem cells are mesenchymal stem cells.

[0022] In some embodiments, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

[0023] In some embodiments, the diameter of the vesicle is 0.03-10 μm. In some embodiments, the diameter of the vesicle is 0.03-6 μm. In some embodiments, the diameter of the vesicle is 0.03-4.5 μm. In some embodiments, the diameter of the vesicle is 0.03-1 μm. In some embodiments, the concentration of staurosporine is 1-15000 nM. In some embodiments, the concentration of staurosporine is 200-10000 nM. In some embodiments, the concentration of staurosporine is 500-1000 nM. In some embodiments, the concentration of staurosporine is 500-900 nM. In some embodiments, the concentration of staurosporine is 500-800 nM.

[0024] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 1 to 900 nM.

[0025] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 10 to 800 nM.

[0026] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 100 to 800 nM.

[0027] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 200 to 700 nM.

[0028] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 300-600 nM.

[0029] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 400-600 nM.

[0030] In some embodiments, the expression of the marker Syntaxin 4 by the vesicles is higher than that by MSCs or exosomes from the same source.

[0031] In some embodiments, the markers further include one or more of Annexin V, Flotillin-1, Cadherin 11 or Integrin alpha 5.

[0032] In some embodiments, the vesicles highly express markers Annexin V, Flotillin-1, Cadherin 11 or Integrin alpha 5.

[0033] In some embodiments, the induced vesicles express markers such as Annexin V, Flotillin-1, Cadherin11, and Integrin alpha 5 at higher levels than MSCs or exosomes from the same source.

[0034] In some embodiments, the method for preparing the vesicle comprises the following steps: 1) culturing mesenchymal stem cells; 2) adding the mesenchymal stem cells of step 1) to a serum-free culture medium containing 500-1000 nM staurosporine, and collecting the cell supernatant; 3) subjecting the cell supernatant collected in step 2) to ultracentrifugation separation to obtain the vesicle.

[0035] In some embodiments, the ultracentrifugation method for separating the vesicles comprises: (a) subjecting the collected culture supernatant to a first centrifugation to obtain the supernatant; (b) subjecting the supernatant collected in step (a) to a second centrifugation to obtain the supernatant; (c) subjecting the supernatant received in step (b) to a third centrifugation to obtain the precipitate; (d) subjecting the precipitate received in step (c) to a fourth centrifugation to obtain the precipitate.

[0036] In some embodiments, the first centrifugation is performed at 500-1500 g for 5-30 minutes.

[0037] In some embodiments, the first centrifugation is performed at 500-1000 g for 5-20 minutes.

[0038] In some embodiments, the first centrifugation is performed at 500-900 g for 5-15 minutes.

[0039] In some embodiments, the second centrifugation is performed at 1000-3000 g for 5-30 minutes.

[0040] In some embodiments, the second centrifugation is performed at 1500-2500 g for 5-20 minutes.

[0041] In some embodiments, the second centrifugation is performed at 1500-2200 g for 5-15 minutes.

[0042] In some embodiments, the third centrifugation is performed at 10,000-30,000 g for 15-60 minutes.

[0043] In some embodiments, the third centrifugation is performed at 12000-25000 g for 20-60 minutes.

[0044] In some embodiments, the third centrifugation is performed at 12000-20000 g for 20-40 minutes.

[0045] In some embodiments, the fourth centrifugation is performed at 10,000-30,000 g for 15-60 minutes.

[0046] In some embodiments, the fourth centrifugation is performed at 12000-25000 g for 20-60 minutes.

[0047] In some embodiments, the fourth centrifugation is performed at 12000-20000 g for 20-40 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1ApoVs were shown to be able to produce H2S. (A) Expression of CBS, CSE and 3-MST in mBMSCs, apoptotic mBMSCs (apoMSCs) and apoVs was assessed by Western blotting. (B) Expression of CBS, CSE and 3-MST in apoVs was assessed by immunofluorescence (scale bar: 100 nm). (C) H2S microelectrode measurement showed that apoVs produced H2S, which was inhibited by HA and PAG treatment, n = 5 per group. (D) CBS - / - apoVs and CSE - / - The H2S concentration of apoVs was lower than that of apoVs, n = 5. (E) H2S probe showed that apoVs produced H2S, which was inhibited by HA and PAG treatment. L-cystine can promote H2S production in apoVs, and the arrow indicates H2S. (F) - / - apoVs and CSE - / - apoVs generated less H2S than apoVs, as assessed by the H2S probe. Arrows indicate H2S. *P<0.05; **P<0.01; ***P<0.001.

[0049] Figure 2 Expression of apoVs markers is shown. (A) apoVs-related proteins were analyzed by Western blotting. Compared with apo-mBMSCs, apoVs highly expressed EV-related markers CD63, CD81, TSG101, and Alix. Compared with exosomes, apoVs highly expressed cleaved caspase 3. (B) Western blotting analysis of CBS - / - apoVs and CSE - / - Expression of CBS and CSE in apoVs. (C) Western blot showing knockdown of 3-MST in apoVs by siRNA. (D) H2S microelectrode showing reduced H2S concentration from si-3-MST apoVs compared to apoVs collected from the scrambled control group, n=5 per group. * P < 0.05; ** P < 0.01; *** P<0.001. Scramble group refers to cells transfected with scrambled sequences; si-3-MST group refers to cells transfected with 3-MST homologous sequences; Scramble apoVs refers to apoVs extracted after apoptosis was induced in cells transfected with scrambled sequences; si-3-MSTapoVs refers to apoVs extracted after apoptosis was induced in cells transfected with 3-MST homologous sequences.

[0050] Figure 3 It shows that apoVs depends on CBS and CBE to produce H2S. The H2S probe shows that the H2S from CBS- / - and CSE - / - The level of H2S produced by apoVs induced by mouse mBMSC cultured in vitro was significantly decreased.

[0051] Figure 4 It shows that apoVs depends on CBS and CBE to produce H2S. H2S microelectrode shows that the apoVs group can produce a lot of H2S compared with the blank group, while CBS - / - and CSE - / - The level of H2S produced by apoVs induced by mouse mBMSC cultured in vitro was significantly decreased, n=5 in each group. * P < 0.05; ** P < 0.01; *** P<0.001.

[0052] Figure 5 The results showed that apoVs has an antioxidant effect on skin fibroblasts. The application of apoVs can significantly reduce the oxidative stress level of skin fibroblasts caused by H2O2, but CBS - / - apoVs and CSE - / - The effect of apoVs was weakened, n=3 in each group. * P < 0.05; ** P < 0.01; *** P<0.001.

[0053] Figure 6 The results showed that apoVs has an antioxidant effect on human mesenchymal stem cells. The application of apoVs can significantly reduce the oxidative stress level of human mesenchymal stem cells caused by H2O2, but CBS - / - apoVs and CSE - / - The effect of apoVs was weakened, n=3 in each group. * P < 0.05; ** P < 0.01; *** P<0.001.

[0054] Figure 7 The results showed that apoVs has an antioxidant effect on human vascular endothelial cells. The application of apoVs can significantly reduce the oxidative stress level of human vascular endothelial cells caused by H2O2, but the effect of CBS- / -apoVs and CSE- / -apoVs is weakened. n = 3 in each group. * P < 0.05; ** P < 0.01; *** P<0.001.

[0055] Figure 8It shows that apoVs can inhibit oxidative stress in renal epithelial cells by generating H2S to treat chronic kidney disease. The application of apoVs can significantly reduce the concentration of CREA and BUN in CKD mouse models and increase the glomerular filtration rate, but CBS - / - apoVs and CSE - / - The effect of apoVs was weakened, n=3 in each group. * P < 0.05; ** P < 0.01; *** P<0.001.

[0056] Fig. 9 It was shown that STS induced mBMSCs, PBMCs and CD4 + Flow cytometry showed that (A) STS and (B) UV could induce apoptosis of mBMSCs, PBMCs and CD4 + Apoptosis of T cells, which could be inhibited by pretreatment with Z-VAD, n=3 per group. ** P < 0.01; *** P<0.001.

[0057] Fig.10 Apoptotic cells are shown to release H2S in vitro. (AD) H2S concentrations from different cell supernatants were evaluated using H2S microelectrodes (A and B) and lead acetate formation assays (C and D) after apoptosis induction (STS and UV treatment), n = 5 per group. Both H2S microelectrode results and lead acetate formation assays demonstrated that STS and UV treatment significantly increased H2S concentrations in mBMSCs, PBMCs, and CD4+ T cells, while H2S concentrations were reduced by the addition of Z-VAD (a pan-caspase inhibitor). (E) H2S concentrations at different time points during STS stimulation were detected using H2S microelectrodes. H2S concentrations at 3 and 6 h were higher than those at 9 h of STS treatment, n = 5. (F) STS induced significant H2S production at 3 and 6 h, while H2S production decreased 9 h after induction, scale bar, 20 μm. (G) Expression of H2S-producing enzymes was evaluated by qRT-PCR. STS treatment significantly promoted gene expression of CBS and CSE at 3 and 6 h of STS treatment, n = 3. (H) CBS expression increased at 3, 6 and 9 h of STS treatment. CSE and 3-MST expression increased at 6 and 9 h of STS treatment, n = 3. (I) Green maleimide assay was used to detect sulfhydration in mBMSCs lysates after STS induction. STS treatment can enhance sulfhydration of mBMSCs 3 h after STS treatment, n = 3. NS, not significant. * P < 0.05;

[0058] ** P < 0.01; ***P<0.001. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0060] "Comprising" or "including" is intended to indicate that compositions (e.g., media) and methods include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means excluding other elements that have any significance for the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel features of the claimed application. "Consisting of" means excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present application.

[0061] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition, combination, construction, etc. is described as including (or comprising) components A, B, C, and / or D, the composition can include A alone; B alone; C alone; D alone; A and B in combination; A and C in combination; A and D in combination; B and C in combination; B and D in combination; C and D in combination; or A, B, C, and D in combination. STS in the present invention is staurosporine.

[0062] Herein, "ApoV" is the same as "ApoVs" or "apoVs" or "MSC-ApoV", which refers to "induced vesicles", which can also be referred to as IEVs. IEVs in the embodiments of the present invention are short for induced vesicles, which can be referred to as induced vesicles, or induced extracellular vesicles (Induced extracellular vesicles, IEVs), and IEV is the same as IEVs. Induced extracellular vesicles refer to a type of subcellular product produced when precursor cells (such as stem cells) are intervened or induced to cause apoptosis during normal survival. Usually, this type of subcellular product has a membrane structure, expresses apoptotic markers, and partially contains genetic material DNA. The inventors have found that induced extracellular vesicles are a type of substance that is distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.). In some embodiments, the cells in normal survival are, for example, non-apoptotic cells, non-aging cells, non-aging and proliferation-stagnated cells, non-frozen and revived cells, non-malignant and abnormally proliferating cells, or non-damaged cells. In some embodiments, the cells at normal survival are taken from cells at 80-100% cell contact fusion during cell culture. In some embodiments, the cells at normal survival are taken from logarithmic phase cells. In some embodiments, the cells at normal survival are taken from primary culture and subculture cells derived from human or mouse tissues. In some embodiments, the cells at normal survival are taken from established cell lines or cell strains. In some embodiments, the precursor cells are taken from early cells.

[0063] In this article, EV refers to normal extracellular vesicles (EVs), which are nanometer-sized membrane-structured bodies released by cells during normal culture or under physiological or pathological conditions in vivo. They originate from the endosomal system or fall off from the plasma membrane. Their diameters range from 40 to 1000 nm. They are mainly composed of microvesicles (MV), apoptotic vesicles and exosomes, and contain a variety of signal molecules such as RNA and proteins.

[0064] In some embodiments, the surface protein markers of the obtained "induced vesicles" are identified and characterized by the method described in Zhang X, Tang J, Kou X, Huang W, Zhu Y, Jiang Y, Yang K, Li C, Hao M, Qu Y, Ma L, Chen C, Shi S, Zhou Y. Proteomic analysis of MSC-derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions. J Extracell Vesicles. 2022Jun;11(7):e12240.doi:10.1002 / jev2.12240.PMID:36856683;PMCID:PMC9927920., suggesting that the characteristic markers are consistent with the description in the aforementioned article.

[0065] In some embodiments, the inducible vesicle is as disclosed in patent application 202110077486.9. Patent application 202110077486.9 is incorporated herein by reference in its entirety.

[0066] Among them, hydroxylamine hydrochloride (HA; CBS inhibitor) and propargylglycine (PAG; CSE inhibitor) can block H2S production. Among them, Z-VAD refers to a pan-caspase inhibitor. In this article, all animal experiments were approved by the Animal Ethics Welfare Committee of SunYat-Sen University (SYSU-IACUC-2023-000441).

[0067] In the embodiments herein, CBS - / - mBMSCs and CSE - / - mBMSCs are derived from CBS - / - Mice and CSE - / - Mouse, CBS - / - apoVs and CSE - / - apoVs refers to the CBS - / - mBMSCs and CSE - / - apoVs of mBMSCs.

[0068] Materials and methods

[0069] A H2S microelectrode detection method :

[0070] Cell supernatants and blood were collected into a temperature-controlled microrespiration chamber (Unisense, Aarhs, Denmark), and H2S concentrations were measured by an H2S microelectrode sensor (model H2S-MRCh; Unisense) coupled to a Unisense PA2000 amplifier.

[0071] A lead sulfide detection method :

[0072] H2S from tissues and blood was detected using the lead acetate / lead sulfide method. Briefly, fresh tissues were collected and homogenized. Protein concentration was analyzed using the BCA assay. Then, a reaction mixture was prepared using L-cysteine ​​(10 mM) and 5'-pyridoxal phosphate hydrate (PLP; 1 mM). Protein (100 mg) from each sample or plasma (20 μL) was added to the reaction mixture in a 96-well plate. The plate was covered with lead acetate paper and incubated at 37°C until lead acetate was detected.

[0073] H2S probe staining:

[0074] Endogenous H2S production of cells and apoV was detected by Washington State Probe-5 (WSP-5), which is based on the dinucleophilicity of H2S and selectively reacts with H2S to produce a green fluorescent signal. 50 Cells and apoV were incubated with WSP-5 for 30 minutes and washed three times with PBS. The fluorescence of WSP-5 was detected by confocal microscopy.

[0075] Western blotting :

[0076] Co-IP assays were performed using a Pierce MS-compatible magnetic IP kit. Briefly, cell lysates were incubated with antibodies overnight at 4°C, followed by incubation with protein A / G magnetic beads for 2 h, and after washing five times, the eluted precipitate was used for immunoblotting and mass spectrometry detection. Western blotting was performed as described previously, and 47 total proteins were extracted using a total protein extraction kit (ThermoFisher) according to the manufacturer's protocol. Nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Beijing, China). For western blotting, 20 μg of protein was used for electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking for 1 h, the membrane was incubated with the corresponding primary antibody overnight at 4°C and with an HRP-conjugated secondary antibody for 1 h. Finally, the membrane was visualized by SuperSignal West Pico Chemimetric Matter (Thermo Fisher) and visualized by ChemiDoc TM The samples were scanned using the MP imaging system (Bio-Rad, CA, USA).

[0077] Quantitative PCR analysis :

[0078] Total RNA was extracted from cells and tissues using TRIzol reagent (Life Technologies, MD, USA), and cDNA was synthesized using the Reverse Transcriptase M-MLV Kit (TaKaRa, Tokyo, Japan). Gene expression levels were quantified by RT-PCR using the SYBR Green kit (Roche, Basel, Switzerland) with gene-specific primers (Table S2).

[0079] Sulfide hydration assay using maleimide :

[0080] 51 Cell proteins were collected and incubated with Alexa Fluor 488C5 maleimide (10 μM) at 4°C for 2 hours. After washing, primary antibodies were added and incubated overnight at 4°C; IgG was used as a control. Protein A / G magnetic beads were added for 2 hours at 4°C, and immunoprecipitates were collected after centrifugation at 2,500 rpm for 5 minutes at 4°C. Beads were precipitated and washed, then treated with or without DTT (1 mM) for 1 hour at 4°C after boiling. Samples were used for electrophoresis and transferred to PVDF membranes using ChemiDoc TM MP Imaging System scanning PVDF membrane.

[0081] Statistical analysis: Normal distribution was assessed using the Shapiro-Wilk test. Comparisons between two groups were analyzed using independent unpaired two-tailed Student's t-tests to obtain normally distributed data. Comparisons between more than two groups were analyzed using one-way ANOVA with Tukey's test. P values ​​less than 0.05 were considered statistically significant.

[0082] B6.129P2-Cbsm1 Unc / J(CBS - / - , JAX#002461) mice were a gift from Prof. Shi Yinghong (Fudan University). tm1(Luc)Smoc (CSE- / -, NM-KI-00004, Shanghai Model Organisms Technology Co., Ltd., China) mice were a gift from Prof. Bin Geng (Chinese Academy of Medical Sciences).

[0083] In this article, the isolation method of mBMSCs: Bone marrow cells were flushed out from the bone marrow of the femur with 5% fetal bovine serum (FBS; Gibco BRL, NY, USA) in PBS. Single cell suspension was obtained through a 70μm cell strainer (BD Biosciences, CA, USA). The cells were incubated at 37°C in 5% CO2 for 48 hours. Non-adherent cells were washed away with PBS, and mBMSCs were cultured for 10 days with α-MEM medium (Gibco BRL) supplemented with 10% FBS, 2mM L-glutamine (Gibco BRL), 50μM 2-mercaptoethanol (Gibco BRL), 100U / ml penicillin and 100μg / ml streptomycin (Gibco BRL). From CBS, respectively. - / - Mice and CSE - / - Isolation of CBS from mice - / - mBMSCs and CSE - / - mBMSCs.

[0084] Example 1 Obtaining vesicles

[0085] 1. STS induces vesicle production

[0086] mBMSCs were incubated with STS (250 nM) at 37 °C for 10 h to induce apoptosis. Continuous centrifugation (800 g for 10 min, 2,000 g for 10 min, 16,000 g for 30 min, and 16,000 g for 30 min) was performed to separate ApoVs. ApoVs were characterized by transmission electron microscopy (TEM; FEI Company, MA, USA), western blotting, and ZetaView instrument (Particle Metrix, Ammersee, Germany) as previously reported. - / - mBMSCs、CSE - / - Isolation of CBS from mBMSCs and si-3-MSTmBMSCs - / - ApoVs、CSE - / - ApoVs and si-3-MST ApoVs.

[0087] Among them, the method for obtaining ApoVs refers to Chinese patent application 202110077923.7.

[0088] 2. UV-induced vesicle production

[0089] mMBSCs were heated at 150 mJ / cm -2Cells were irradiated under UV-C (Stratalinker) for 6 h to induce apoptosis. Continuous centrifugation (800 g for 10 min, 2,000 g for 10 min, 16,000 g for 30 min, and 16,000 g for 30 min) was performed to separate ApoVs. ApoVs were characterized by transmission electron microscopy (TEM; FEI Company, MA, USA), western blotting, and ZetaView instrument (Particle Metrix, Ammersee, Germany) as previously reported. - / - mBMSCs、CSE - / - Isolation of CBS from mBMSCs using ApoVs and si-3-MST - / - ApoVs、CSE - / - ApoVs and si-3-MSTApoSCs.

[0090] Among them, the method of obtaining ApoVs is different except for the apoptosis induction method, and the rest is referred to Chinese patent application 202110077923.7.

[0091] Example 2 Inducible vesicles produce H2S

[0092] 1. Experimental methods

[0093] 1.1 STS induced vesicle production (same as STS induction method in Example 1)

[0094] mBMSCs were incubated with STS (250 nM) at 37°C for 10 h to induce apoptosis. Continuous centrifugation (800 g for 10 min, 2,000 g for 10 min, 16,000 g for 30 min, 16,000 g for 30 min) was performed to separate ApoVs. ApoVs were characterized by transmission electron microscopy (TEM; FEI Company, MA, USA), western blotting and ZetaView instrument (Particle Metrix, Ammersee, Germany).

[0095] Among them, the steps of the method for obtaining exosomes are as follows: when mBMSCs reach 80% confluence, the cells are washed with PBS and the medium without FBS is replaced. After 40 hours, the culture supernatant is collected and centrifuged at 800g for 10 minutes and 2000g for 10 minutes to remove cell debris. Subsequently, the supernatant is centrifuged at 16000g for 30 minutes to remove medium EVs, and centrifuged at 120000g for 2 hours to obtain exosomes.

[0096] 1.2 Western blotting

[0097] 20 μg of protein was used for electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking for 1 h, the membrane was incubated with the corresponding primary antibody overnight at 4 °C and with HRP-conjugated secondary antibody for 1 h. Finally, the membrane was visualized by chemiluminescent substrate (Thermo Fisher) and analyzed by ChemiDoc TM The samples were scanned using the MP imaging system (Bio-Rad, CA, USA).

[0098] 1.3 Immunofluorescence

[0099] The ApoVs pellet was resuspended in 100 μl PBS, fixed with 900 μl 4% PFA, permeabilized with 0.2% Triton-X, centrifuged at 16,000 g for 30 min, and the pellet was resuspended as before, incubated with the corresponding primary antibody at room temperature for 1 hour, centrifuged at 16,000 g for 30 min, and the pellet was resuspended as before, incubated with the corresponding secondary antibody at room temperature for 1 hour, and mounted with DAPI-containing mounting medium. Images were collected by super-resolution SIM (Carl Zeiss).

[0100] 1.4H2S microelectrode detection

[0101] Cell supernatants and blood were collected in a temperature-controlled microrespiration chamber (Unisense, Aarhus, Denmark), and H2S concentration was detected by a H2S microelectrode sensor (Model H2S-MRCh; Unisense) and a Unisense PA2000 amplifier.

[0102] 2. Experimental results

[0103] Compared with apo-mBMSCs, apoVs highly expressed EV-related markers CD63, CD81, TSG101, and Alix. Compared with exosomes, apoVs highly expressed cleaved caspase 3 ( Figure 2 A). We found that apoptotic mBMSCs (i.e., mBMSCs group, ApoMSCs) and ApoVs expressed CBS, CSE, and 3-MST ( Figure 1 A and 1B). Microelectrode and confocal microscopy analysis showed that apoVs can produce H2S ( Figure 1 C and 1E). HA or PAG treatment inhibited H2S production ( Figure 1 C and 1E). In addition, we obtained - / - mBMSCs and CSE - / - mBMSCs collected ApoVs and named them CBS - / - ApoVs and CSE - / - ApoVs( Figure 2 B).

[0104] Microelectrodes and confocal microscopy analysis revealed that CBS - / - apoVs and CSE - / - The H2S concentration of apoVs was lower than that of apoVs( Figure 1 D and 1F).

[0105] In addition, siRNA was used to knock down 3-MST levels in mBMSCs, and si-3-MST apoVs ( Figure 2 C). The results showed that the H2S level in si-3-MST apovs was lower than that in apovs ( Figure 2 D). Therefore, these studies have proved that apoV has the ability to produce H2S. At the same time, we verified that the production of H2S by apoptotic vesicles also depends on two key enzymes for H2S production in cells, CBS and CSE ( Figure 3 and Figure 4 ).

[0106] in, Figure 2 CBS - / - mBMSCs and CSE - / - mBMSCs are derived from CBS - / - Mice and CSE - / - Mouse, Figure 1 CBS - / - apoVs and CSE - / - apoVs refers to the CBS derived from - / - mBMSCs and CSE - / - apoVs of mBMSCs.

[0107] ApoMSCs refers to MSCs that have been induced to apoptosis.

[0108] The scramble group refers to cells transfected with scrambled sequences; the si-3-MST group refers to cells transfected with 3-MST homologous sequences; Scramble apoVs refers to apoVs extracted from cells transfected with scrambled sequences and induced apoptosis; si-3-MST apoVs refers to apoVs extracted from cells transfected with 3-MST homologous sequences and induced apoptosis. All other conditions were the same for each group unless otherwise specified.

[0109] Example 3 Antioxidant effect of inducible vesicles on skin fibroblasts

[0110] 1. Isolation and culture method of skin fibroblasts

[0111] 1.1 Reagents:

[0112] Tissue digestion solution (DMEM medium + 1 mg / ml collagenase + 1 mg / ml DNase I) PBS, DEME medium, FBS, double antibody (penicillin-streptomycin), alcohol, red blood cell lysis solution (optional) [To increase the separation efficiency, type I and type IV collagenase are sometimes used in combination.

[0113] 1.2 Tissue separation:

[0114] 1.2.1. After obtaining the surgical specimen, soak it in serum-free DMEM medium + 0.1% double antibody as soon as possible, place it on ice, and bring it back to the laboratory for processing as soon as possible (DMEM medium should be prepared in advance, and it should not contain serum. Prepare EP tubes and culture medium according to the number of patients, place them in an ice box, and then go to the hospital to take samples).

[0115] 1.2.2. Rapidly rinse the specimen with alcohol (3 seconds), then soak it in pre-cooled PBS. In this step, use a culture dish to hold alcohol and PBS respectively.

[0116] 1.2.3. Use scissors to remove the burnt skin tissue and fat tissue, and cut the tissue into small pieces (mm). You can add a little digestive solution to facilitate cutting. If 1ml digestive solution is added, transfer the cut tissue and digestive solution to a 1.5ml EP tube, and then use spring scissors to continue to cut the tissue as small as possible. If there are many tissue pieces, transfer multiple tubes of tissue and digestive solution to a 15ml EP tube, seal it with sealing film, and put it in a constant temperature incubator to rotate and fix it.

[0117] 1.2.4.Digest at 37℃, 35rpm for 1h, and observe the tissue digestion during the process.

[0118] 1.2.5. After digestion is completed, blow the tissue vigorously with the Pasteur tube until the tissue is no longer blocked. If blowing is difficult, place it in the incubator for 5 minutes and continue blowing. Then change to a 1000ul pipette tip for blowing. After it is smooth, change to a 200ul pipette tip for blowing. If blowing is difficult, place it in the incubator for 5 minutes and continue blowing.

[0119] 1.2.6. Add 1-2 ml of culture medium, centrifuge at 1500 rpm for 5 min, discard the supernatant, add 1 ml of complete culture medium (DMEM culture medium, 10% FBS, 100 U / ml double antibody (penicillin-streptomycin)) to resuspend the precipitate.

[0120] 1.2.7. Filter the cell suspension through a 70um filter and collect all the liquid in the same 50ml tube (when there are too many tissue pieces, add 0.2mg / ml type I collagenase-DMEM and digest overnight. After about 12h, filter with a 70um filter membrane, centrifuge at 1500rpm for 5min, discard the supernatant, add 1ml complete culture medium (DMEM culture medium, 10% FBS, 100U / ml double antibody (penicillin-streptomycin)), resuspend, and transfer to a 10cm culture dish for culture.

[0121] 1.2.8. Take an appropriate amount of cells and spread them in a 10 cm culture dish, adding 8 ml of culture medium to each well.

[0122] 1.2.9. Culture in a 37℃ incubator. Change the medium when a large number of long strip-shaped fibroblasts appear on the bottom. Change the medium every other day. When the cells are full in the 10 cm culture dish, change the medium. Collect the supernatant after 48 hours, subculture at a 1:2 ratio, and use the 4th to 8th generation cells for antioxidant experiments.

[0123] 2. Antioxidant test

[0124] 2.1 Experimental methods

[0125] The cells were cultured to the 4th to 8th generation. When the cells reached 80% confluence, they were divided into H2O2 group, H2O2+apoVs group, H2O2+CBS group, and - / - apoVs group, H2O2+CSE - / - apoVs group, n = 3. According to the experimental requirements, H2O2 (H2O2 100 μMol / L), apoVs (25 μg / ml), CBS - / - apoVs (25 μg / ml) and CSE - / - apoVs (25 μg / ml), and after 4 hours of culture, the cellular oxidative stress level was detected according to the following steps. Among them, the H2O2 group refers to the model group; the H2O2+apoVs group refers to the model group with apoVs added; the H2O2+CBS group refers to the model group with apoVs added; - / - The apoVs group refers to the model group with CBS added - / - apoVs;H2O2+CSE - / - The apoVs group refers to the model group with CSE added - / - apoVs.

[0126] Among them, apoVs, CBS - / - apoVs or CSE - / - The method for obtaining apoVs is the same as the method for STS-induced vesicle production in Example 1.

[0127] ROS detection was performed using a ROS reactive oxygen species detection kit (Solebo). The specific method was as follows: dilute the mother solution with PBS or serum-free culture medium to the required working concentration, about 5-20 μM, before use, incubate at 37°C for 30-40 min, remove the staining solution, rinse twice with 1xPBS, and then detect under a microscope.

[0128] Superoxide dismutase (SOD) activity detection kit (Solebao), malondialdehyde (MDA) content detection kit (Solebao), and glutathione peroxidase (GSH-Px / GPX) activity detection kit (Solebao) were used for SOD, MDA, and GPx detection.

[0129] The specific method is as follows: collect cells into a centrifuge tube, discard the supernatant after centrifugation; add 1 mL of extract for every 5 million cells, and disrupt the cells by ultrasonic wave (power 200W, ultrasonic wave 3s, interval 10s, repeat 30 times); centrifuge at 8000g 4℃ for 10min, take the supernatant, and place it on ice for testing. Dilute the 80μmol / mL standard solution with diluent to a 0.08μmol / mL standard solution, and prepare it before use. After adding the corresponding reagent, mix thoroughly, centrifuge at 4000rpm at room temperature for 5min, take the supernatant to a 96-well plate, and detect it with an enzyme marker.

[0130] 2.2 Experimental Results

[0131] The results are as follows Figure 5 The results showed that apoVs could significantly reduce the oxidative stress level of skin fibroblasts, while CBS - / - apoVs and CSE - / - The effect of apoVs was weakened, indicating that apoVs exerts an antioxidant effect on skin fibroblasts by releasing H2S.

[0132] Example 4 Antioxidant effect of induced vesicles on human mesenchymal stem cells

[0133] 1. Isolation and culture methods of human mesenchymal stem cells

[0134] 1.1 Reagents

[0135] Percoll cell separation solution, FBS, DEME medium, FBS, double antibody (penicillin-streptomycin), alcohol, red blood cell lysis solution (optional).

[0136] 1.2 Cell isolation and culture

[0137] According to the standard bone marrow puncture procedure, 5-10 mL of bone marrow was extracted from the posterior superior iliac spine of voluntary donors, anticoagulated with heparin, diluted with an equal amount of PBS, centrifuged at 900 g for 10 min at room temperature, twice, and the fat and supernatant were discarded. PBS was added to make 4×10 75 mL of the suspension was added to 5 mL of Percoll separation solution, centrifuged at 900 g for 30 min, the middle cell layer was added with 2 times the amount of PBS, centrifuged at 900 g for 5 min, the supernatant was discarded, and 1 × 10 6 / mL density was inoculated in a culture dish, 10mL of complete medium was added, and cultured in a 37℃ incubator. The medium was changed once every 24h and 48h of primary culture. After 7d of culture, the cells were digested with 0.25% trypsin and continued to be cultured at a 1:2 ratio. The cells of the 4th to 8th generations were used for antioxidant experiments.

[0138] The components of the complete culture medium here are the same as those of the complete culture medium in Example 3.

[0139] 2. Antioxidant test

[0140] 2.1 Experimental methods

[0141] The cells were cultured to the 4th to 8th generation. When the cells reached 80% confluence, they were divided into H2O2 group, H2O2+apoVs group, H2O2+CBS group, and - / - apoVs group, H2O2+CSE - / - apoVs group, n=3. According to the experimental requirements, H2O2 (H2O2 100 μMol / L), apoVs (25 μg / ml), CBS - / - apoVs (25 μg / ml) and CSE - / - apoVs (25 μg / ml), and after 4 hours of culture, the cellular oxidative stress level was detected according to the following steps.

[0142] Among them, the H2O2 group refers to the model group; the H2O2+apoVs group refers to the model group with apoVs added; the H2O2+CBS - / - The apoVs group refers to the model group with CBS added - / - apoVs;H2O2+CSE - / - The apoVs group refers to the model group with CSE added - / - apoVs.

[0143] Among them, apoVs, CBS - / - apoVs or CSE - / - The method for obtaining apoVs is the same as the method for STS-induced vesicle production in Example 1.

[0144] ROS detection was performed using a ROS reactive oxygen species detection kit (Solebo). The specific method was as follows: dilute the mother solution with PBS or serum-free culture medium to the required working concentration, about 5-20 μM, before use, incubate at 37°C for 30-40 min, remove the staining solution, rinse twice with 1xPBS, and then detect under a microscope.

[0145] Superoxide dismutase (SOD) activity detection kit (Solaibao), malondialdehyde (MDA) content detection kit (Solaibao), glutathione peroxidase (GSH-Px / GPX) activity detection kit (Solaibao) were used for SOD, MDA, and GPx detection. The specific method is as follows: collect cells into a centrifuge tube, discard the supernatant after centrifugation; add 1 mL of extract solution for every 5 million cells, and ultrasonically disrupt the cells (power 200W, ultrasonic 3s, interval 10s, repeat 30 times); centrifuge at 8000g 4℃ for 10min, take the supernatant, and place it on ice for testing. Dilute the 80μmol / mL standard solution with diluent to a 0.08μmol / mL standard solution, and prepare it before use. After adding the corresponding reagent, mix thoroughly, centrifuge at 4000rpm at room temperature for 5min, take the supernatant to a 96-well plate, and detect it with an enzyme marker.

[0146] 2.2 Experimental Results

[0147] The results are as follows Figure 6 The results showed that apoVs could significantly reduce the oxidative stress level of human mesenchymal stem cells, while CBS - / - The effects of apoVs and CSE- / -apoVs were weakened, indicating that apoVs exerts an antioxidant effect on human mesenchymal stem cells by releasing H2S.

[0148] Example 5 Antioxidant effect of inducible vesicles on human vascular endothelial cells

[0149] 1. Isolation and culture method of human vascular endothelial cells

[0150] 1.1 Reagents:

[0151] Tissue digestion solution (DMEM medium + 1 mg / ml collagenase + 1 mg / ml DNase I), PBS, DEME medium, FBS, double antibody (penicillin-streptomycin), alcohol, red blood cell lysis solution (optional) [To increase the separation efficiency, type I and type IV collagenase are sometimes used together.

[0152] 1.2 Tissue separation:

[0153] 1.2.1 Clamp both ends of the umbilical cord with sterile hemostats, cut the umbilical cord outside the hemostats, and immerse it in 4℃ PBS. The umbilical cord should not be kept at 4℃ for more than 24 hours.

[0154] 1.2.2 Wear sterile gloves, remove the umbilical cord, quickly rinse the specimen with alcohol (3 seconds), and then soak it in pre-cooled PBS. In this step, use a culture dish to hold alcohol and PBS respectively. After disinfecting the inside of the hemostatic forceps with iodine and alcohol cotton balls, use sterile scissors to cut off the two ends of the umbilical cord.

[0155] 1.2.3 Find the vein at one end of the umbilical cord, insert the umbilical cord vein cannula, tie it tightly with two thick silk threads, and use a syringe to draw 30ml of PBS to flush the venous cavity until the umbilical cord blood is washed away.

[0156] 1.2.4 Expel the residual fluid in the venous cavity, clamp the other end of the umbilical cord with a hemostatic forceps, connect the needle to a 10ml syringe, inject about 6-8ml of 37℃ preheated tissue digestion solution, and place it in 37℃ PBS to keep warm for 6-10 minutes.

[0157] 1.2.5 Aspirate the cell suspension digested by the tissue digestion fluid in the venous cavity, add it to a centrifuge tube pre-added with 20 ml PBS, rinse the venous cavity, and add it to the centrifuge tube.

[0158] 1.2.6 Centrifuge at 1500 rpm for 10 min, discard the supernatant, add 1 ml of complete culture medium (DMEM culture medium, 10% FBS, 100 U / ml double antibody (penicillin-streptomycin)) and resuspend the precipitate.

[0159] 1.2.7 1×10 6 / mL density was inoculated in a culture dish, 10mL of complete culture medium was added, and cultured in a 37℃ incubator. The primary culture medium was changed once every 24h and 48h. After 7d of culture, 0.25% trypsin was used for digestion, and the culture was continued at a 1:2 ratio. 2. Antioxidant experiment

[0160] 2.1 Experimental methods

[0161] The cells were cultured to the 4th to 8th generation. When the cells reached 80% confluence, they were divided into H2O2 group, H2O2+apoVs group, H2O2+CBS group, and - / - apoVs group, H2O2+CSE - / - apoVs group, n=3. According to the experimental requirements, H2O2 (H2O2 100 μMol / L), apoVs (25 μg / ml), CBS - / - apoVs (25 μg / ml), CSE - / - apoVs (25 μg / ml), and after 4 hours of culture, the cellular oxidative stress level was detected according to the following steps.

[0162] Among them, the H2O2 group refers to the model group; the H2O2+apoVs group refers to the model group with apoVs added; the H2O2+CBS - / - The apoVs group refers to the model group with CBS added - / - apoVs;H2O2+CSE - / - The apoVs group refers to the model group with CSE added - / - apoVs. Among them, apoVs, CBS - / - apoVs or CSE- / - The method for obtaining apoVs is the same as the method for STS-induced vesicle production in Example 1.

[0163] ROS detection was performed using a ROS reactive oxygen species detection kit (Solebo). The specific method was as follows: dilute the mother solution with PBS or serum-free culture medium to the required working concentration, about 5-20 μM, before use, incubate at 37°C for 30-40 min, remove the staining solution, rinse twice with 1xPBS, and then detect under a microscope.

[0164] Superoxide dismutase (SOD) activity detection kit (Solaibao), malondialdehyde (MDA) content detection kit (Solaibao), glutathione peroxidase (GSH-Px / GPX) activity detection kit (Solaibao) were used for SOD, MDA, and GPx detection. The specific method is as follows: collect cells into a centrifuge tube, discard the supernatant after centrifugation; add 1 mL of extract solution for every 5 million cells, and ultrasonically disrupt the cells (power 200W, ultrasonic 3s, interval 10s, repeat 30 times); centrifuge at 8000g 4℃ for 10min, take the supernatant, and place it on ice for testing. Dilute the 80μmol / mL standard solution with diluent to a 0.08μmol / mL standard solution, and prepare it before use. After adding the corresponding reagent, mix thoroughly, centrifuge at 4000rpm at room temperature for 5min, take the supernatant to a 96-well plate, and detect it with an enzyme marker.

[0165] 2.2 Experimental Results

[0166] The results are as follows Figure 7 The results showed that apoVs could significantly reduce the oxidative stress level of human vascular cells, while CBS - / - apoVs and CSE - / - The effect of apoVs was weakened, indicating that apoVs exerts an antioxidant effect on human vascular endothelial cells by releasing H2S.

[0167] Example 6 Inducible vesicles inhibit oxidative stress in renal epithelial cells to treat chronic kidney disease by generating H2S

[0168] 1. Experimental methods

[0169] 1.1 Chronic Kidney Disease Model (CKD)

[0170] After the mice were adapted to the environment for one week, the mice in the control group were fed a normal diet, and the mice in the model group were fed a normal diet supplemented with 0.2% adenine for 8 weeks, after which their renal function was evaluated.

[0171] 1.2 ApoVs for the treatment of chronic kidney disease

[0172] The mice were divided into control group, CKD group, CKD+apoVs group, CKD+CBS group - / -apoVs group and CKD+CSE - / - apoVs group, n = 3. apoVs (1×10 6 ), CBS - / - apoVs(1×10 6 ), CSE - / - apoVs(1×10 6 Each of the 100 μl samples was resuspended in 200 μl PBS and injected into the tail vein according to the experimental requirements. After one week, renal function was evaluated as described below.

[0173] The control group refers to the control group mice with a normal diet; the CKD group refers to the model group mice; the CKD+apoVs group refers to the model group mice treated with apoVs; and the CKD+CBS group refers to the model group mice treated with apoVs. - / - The apoVs group refers to the model group mice given CBS - / - apoVs treatment; CKD+CSE - / - The apoVs group refers to the model group mice given CSE - / - apoVs treatment.

[0174] Among them, apoVs, CBS - / - apoVs or CSE - / - The method for obtaining apoVs is the same as the method for STS-induced vesicle production in Example 1.

[0175] 1.3 Assessment of renal function

[0176] Urea nitrogen (BUN) test kit (Nanjing Jiancheng) was used to measure the BUN and CREA levels of mice. The specific method is as follows: EDTA anticoagulated plasma was obtained by enucleation of mice, and the standard application solution was prepared according to the instructions. Blank tube, standard tube and measurement tube were set up, and 0.25 ml of buffered enzyme solution was added. Then, 0.02 ml of double distilled water, 0.02 ml of 10 mmol / L BUN standard application solution and 0.02 ml of the sample to be tested were added in sequence; mixed, and bathed in 37°C water for 10 min, 1 ml of phenol color developer and 1 ml of alkaline sodium hypochlorite were added, mixed thoroughly, and bathed in 37°C water for 10 min, with a wavelength of 640 nm and a light path of 1 cm. The absorbance OD value of each tube was measured.

[0177] Creatinine (Cr) assay kit (Nanjing Jiancheng), EDTA anticoagulated plasma was obtained by mouse enucleation method, blank tube, standard tube and assay tube were set up, 180 μL of enzyme solution A was added, then 6 μL of double distilled water, 6 μL of standard substance and 6 μL of sample were added in sequence, incubated at 37°C for 5 minutes, and the absorbance value A1 was measured at a wavelength of 546 nm. 60 μL of enzyme solution B was added, incubated at 37°C for 5 minutes, and the absorbance value A1 was measured at a wavelength of 546 nm. ΔA=A2-K*A1, K is the dilution factor.

[0178] Creatinine is a metabolite of nitrogen-containing creatine in skeletal muscle cells that is freely filtered by the glomerulus and is not reabsorbed. Therefore, creatinine clearance is used to reflect the glomerular filtration rate. Glomerular filtration rate = urine creatinine concentration × 24h urine volume / serum creatinine concentration.

[0179] 2. Experimental results

[0180] The results are as follows Figure 8 The results showed that apoVs can treat chronic kidney disease in mice, but CBS - / - apoVs and CSE - / - The therapeutic effect of apoVs was weakened.

[0181] Example 7: Apoptotic cells can produce H2S

[0182] 1. Experimental methods

[0183] 1.1 Induction of apoptosis

[0184] mBMSCs were incubated with STS (250 nM) at 37°C for 10 h, PBMCs and CD4+ T cells were incubated with STS (250 nM) at 37°C for 6 h; or mMBSCs were incubated with STS (150 mJ / cm -2 PBMCs and CD4+ T cells were irradiated with UV-C (Stratalinker) for 6 hours at 150 mJ / cm -2 The cells were irradiated with UV-C (Stratalinker) for 4 h to induce apoptosis.

[0185] 1.2 Flow cytometry

[0186] For apoptosis rate determination, cells were stained with FITC anti-Annexin V and PE-Cy7 anti-7AAD for 15 min at room temperature. Cells were then analyzed by flow cytometry (NovoCyte, Agilent Technologies, CA, USA).

[0187] 1.3H2S microelectrode detection

[0188] Cell supernatants and blood were collected in a temperature-controlled microrespiration chamber (Unisense, Aarhus, Denmark), and H2S concentration was detected by a H2S microelectrode sensor (Model H2S-MRCh; Unisense) and a Unisense PA2000 amplifier.

[0189] 1.4 Lead sulfide detection method

[0190] H2S in tissue and blood was detected using the lead acetate / lead sulfide method. Briefly, fresh tissues were collected and homogenized. Protein concentration was analyzed using the BCA assay. Then, a reaction mixture was prepared with L-cysteine ​​(10 mM) and pyridoxal 5′-phosphate hydrate (PLP; 1 mM). Protein (100 mg) or plasma (20 μL) from each sample was added to the reaction mixture in a 96-well plate. The plate was covered with lead acetate paper and incubated at 37°C until lead acetate was detected.

[0191] 1.5 H2S probe staining

[0192] Endogenous H2S production of cells and apoV was detected by Washington State Probe-5 (WSP-5), which is based on the dinucleophilicity of H2S and selectively reacts with H2S to produce a green fluorescent signal. 50 Cells and apoV were incubated with WSP-5 for 30 minutes and washed three times with PBS. The fluorescence of WSP-5 was detected by confocal microscopy.

[0193] 1.6 Western blotting

[0194] Co-IP assays were performed using a Pierce MS-compatible magnetic IP kit. Briefly, cell lysates were incubated with antibodies overnight at 4°C, followed by incubation with protein A / G magnetic beads for 2 h, and after washing five times, the eluted precipitate was used for immunoblotting and mass spectrometry detection. Western blotting was performed as described previously, and 47 total proteins were extracted using a total protein extraction kit (ThermoFisher) according to the manufacturer's protocol. Nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Beijing, China). For western blotting, 20 μg of protein was used for electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking for 1 h, the membrane was incubated with the corresponding primary antibody overnight at 4°C and with an HRP-conjugated secondary antibody for 1 h. Finally, the membrane was visualized by SuperSignal West Pico Chemimetric Matter (Thermo Fisher) and visualized by ChemiDoc TM The samples were scanned using the MP imaging system (Bio-Rad, CA, USA).

[0195] 1.7 Quantitative PCR analysis :

[0196] Total RNA was extracted from cells and tissues using TRIzol reagent (Life Technologies, MD, USA), and cDNA was synthesized using the Reverse Transcriptase M-MLV Kit (TaKaRa, Tokyo, Japan). Gene expression levels were quantified by RT-PCR using the SYBR Green kit (Roche, Basel, Switzerland) with gene-specific primers (Table S2).

[0197] 1.8 Sulfurization and hydration assay using maleimide :

[0198] Cellular proteins were collected and incubated with Alexa Fluor 488C5 maleimide (10 μM) at 4°C for 2 hours, washed, and then primary antibodies were added and incubated overnight at 4°C; IgG was used as a control. Protein A / G magnetic beads were added for 2 hours at 4°C, and immunoprecipitates were collected after centrifugation at 2,500 rpm for 5 minutes at 4°C. Beads were precipitated and washed, then treated with or without DTT (1 mM) at 4°C for 1 hour after boiling, and samples were used for electrophoresis and transferred to PVDF membranes using ChemiDoc TM MP Imaging System scanning PVDF membrane.

[0199] 2. Experimental results

[0200] STS and ultraviolet (UV) irradiation were used to induce apoptosis in mouse bone marrow stem cells (mBMSCs), PBMCs, and CD4+ T cells. Z-VAD was used to inhibit the apoptotic process ( Fig. 9 A and 9B). Microelectrode experiments demonstrated that STS- and UV-induced apoptotic cells showed elevated H2S levels in the cell supernatant ( Fig.10 A and 10B). Lead acetate paper analysis demonstrated that STS-induced cell apoptosis could increase the H2S level in the cell supernatant ( Fig.10 C and 10D). However, pretreatment with Z-VAD reduced the level of H2S in the supernatant of apoptotic cells ( Fig.10 A-10D).

[0201] To further analyze the production of H2S in apoptotic cells, we used STS-induced apoptotic mouse BMSCs (mBMSCs) as a model and selected 3, 6, and 9 hours after STS treatment, which was defined as early, middle, and late apoptosis. H2S probe staining showed that STS treatment produced H2S microelectrodes in early and middle apoptosis compared with control cells ( Fig.10 E and 10F). During apoptosis, the expression of CBS increased significantly, while the expression of CSE and 3-MST increased slightly ( Fig.10 G and 10H). In addition, the thiolation level increased significantly in the early and middle stages of apoptosis ( Fig.10 I). These results indicate that apoptotic mBMSCs mainly produce H2S in the early and middle stages of apoptosis.

Claims

1. Application of inducible vesicles in the preparation of antioxidant products.

2. The use according to claim 1, characterized in that The vesicles are vesicles produced by inducing apoptosis of stem cells by external factors when the stem cells are in normal survival; Preferably, the induction method comprises adding staurosporine, ultraviolet irradiation, starvation method, or heat stress method; Preferably, the stem cells are mesenchymal stem cells; Preferably, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

3. The use according to claim 1, characterized in that The products include medicines, foods, health products, skin care products, or medical and aesthetic products; Preferably, the anti-oxidation comprises reducing the MDA content or reducing the ROS activity, or increasing the SOD activity or increasing the glutathione peroxidase activity; Preferably, the antioxidant comprises antioxidant for cells; Preferably, the cells include skin fibroblasts, stem cells, or vascular endothelial cells; Preferably, the stem cells include mesenchymal stem cells; Preferably, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

4. The use according to claim 1, characterized in that The diameter of the vesicle is 0.03-10 μm; Preferably, the diameter of the vesicle is 0.03-6 μm; Preferably, the diameter of the vesicle is 0.03-4.5 μm; Preferably, the diameter of the vesicle is 0.03-1 μm. Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 1 to 900 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 10 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 100 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 200 to 700 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 300-600 nM; Preferably, cell apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 400-600 nM.

5. The use according to any one of claims 1 to 4, characterized in that: The vesicles express a marker, Syntaxin 4, higher than MSCs or exosomes from the same source; Preferably, the markers further include one or more of Annexin V, Flotillin-1, Cadherin 11 or Integrin alpha5; Preferably, the vesicles highly express the markers Annexin V, Flotillin-1, Cadherin 11 or Integrin alpha 5; preferably, the inducible vesicles express the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 at a higher level than MSCs or exosomes of the same origin; Preferably, the inducible vesicles are antioxidant by producing hydrogen sulfide.

6. Application of induced vesicles in the preparation of drugs for the treatment of chronic kidney disease.

7. The use according to claim 6, characterized in that The chronic kidney disease includes chronic nephritis; Preferably, the chronic kidney disease is selected from glomerulonephritis; Preferably, the vesicles are vesicles produced by inducing apoptosis of stem cells by external factors when the stem cells are in normal survival; Preferably, the induction method comprises adding staurosporine, ultraviolet irradiation, starvation method, or heat stress method; Preferably, the stem cells are mesenchymal stem cells; Preferably, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

8. The use according to claim 6, characterized in that The diameter of the vesicle is 0.03-10 μm; Preferably, the diameter of the vesicle is 0.03-6 μm; Preferably, the diameter of the vesicle is 0.03-4.5 μm; Preferably, the diameter of the vesicle is 0.03-1 μm. Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 1 to 900 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 10 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 100 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 200 to 700 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 300-600 nM; Preferably, cell apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 400-600 nM.

9. The use according to claim 6, characterized in that The vesicles express a marker, Syntaxin 4, higher than MSCs or exosomes from the same source; Preferably, the markers further include one or more of Annexin V, Flotillin-1, Cadherin 11 or Integrin alpha5; Preferably, the vesicles highly express the markers Annexin V, Flotillin-1, Cadherin 11 or Integrin alpha 5; preferably, the inducible vesicles express the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 at a higher level than MSCs or exosomes of the same origin; Preferably, the inducible vesicles are used to treat chronic kidney disease by producing hydrogen sulfide.

10. Application of hydrogen sulfide promoter in the preparation of inducible vesicles for the treatment of antioxidant or chronic kidney disease; Preferably, the accelerator comprises L-cystine; Preferably, the chronic kidney disease is chronic nephritis; Preferably, the chronic kidney disease is glomerulonephritis.

Images