A recombinant human PHB1 protein and its applications

Abstract

This invention discloses a recombinant human PHB1 protein and its applications, belonging to the field of biomedical technology. The amino acid sequence of the recombinant human PHB1 protein is shown in SEQ ID NO.1. In a palmitic acid-induced diabetic cardiomyopathy (DCM) cell model, the recombinant human PHB1 protein significantly reduces the level of reactive oxygen species in cardiomyocytes and decreases the expression of NLRP3 inflammasomes and active Caspase3 protein. Simultaneously, in a DCM mouse model, tail vein injection of recombinant human PHB1 inhibits cardiomyocyte apoptosis and reduces NLRP3 inflammasome expression and TNF-α synthesis in the animal heart. PHB1 is a human protein with no obvious toxic side effects and high safety; theoretically, it has low immune rejection, providing a new and effective drug for the clinical treatment of DCM, and possessing significant clinical application value and industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant human PHB1 protein and its application in the treatment of diabetic cardiomyopathy. Background Technology

[0002] Diabetic cardiomyopathy (DCM) is a unique cardiac complication in diabetic patients, independent of other cardiovascular diseases such as coronary heart disease and hypertension. Its core pathological features include myocardial diastolic / systolic dysfunction, myocardial fibrosis, cardiomyocyte apoptosis, and mitochondrial dysfunction. It can ultimately progress to heart failure and is one of the leading causes of death in diabetic patients. Statistics show that the prevalence of heart failure in diabetic patients is as high as 22%, and the incidence continues to rise. Currently, there are no specific drugs targeting the pathological mechanisms of DCM. Existing treatments mainly focus on controlling blood sugar and improving cardiac function symptomatically, but they cannot reverse myocardial damage, resulting in limited therapeutic effects and an urgent clinical need for treatment.

[0003] Prohibitin 1 (PHB1) is a highly conserved multifunctional protein, primarily located in the inner mitochondrial membrane. It is a key component in maintaining mitochondrial respiratory chain stability and regulating mitochondrial oxidative phosphorylation (OXPHOS) activity, while also participating in the regulation of apoptosis and inflammatory responses. Previous studies have shown that PHB1 function varies depending on its subcellular location. In mitochondria, it regulates mitochondrial fusion and division by stabilizing OPA1 and DRP1, thereby maintaining mitochondrial functional integrity. Previous research has found that PHB1 expression levels are significantly downregulated in STZ-induced DCM animal models and high glucose / palmitate-induced cardiomyocyte models, suggesting that PHB1 may be involved in the pathological process of DCM.

[0004] However, current research on PHB1 primarily focuses on the functional mechanisms of endogenous PHB1, with no reports mentioning that exogenous supplementation of recombinant human PHB1 protein can directly act on cardiomyocytes and improve the pathological damage of diabetic fibrosis (DCM). Furthermore, current technologies do not clearly demonstrate that PHB1 protein can treat DCM by regulating myocardial mitochondrial ATP production, inhibiting myocardial fibrosis, and inhibiting apoptosis, and no publicly disclosed drug compositions containing recombinant human PHB1 protein for DCM treatment have been developed. Therefore, developing novel uses for recombinant human PHB1 protein in DCM treatment, filling existing technological gaps, has significant clinical value and application prospects. Summary of the Invention

[0005] Technical problems solved: In view of the lack of specific treatment drugs for diabetic cardiomyopathy and the lack of reports on the application of exogenous PHB1 protein in the treatment of DCM, this invention provides a recombinant human PHB1 protein and its application, clarifies its mechanism of action, and provides a new target and treatment plan for the clinical treatment of DCM.

[0006] Technical solution: In a first aspect, the present invention provides a recombinant human PHB1 protein, the amino acid sequence of which is shown in SEQ ID NO.1. SEQ ID NO.1: MAAKVFESIGKFGLALAVAGGVVNSALYNVDAGHRAVIFDRFRGVQDIVVGEGTHFLIPWVQKPIIFDCRSRPRNVPVITGSKDLQNVNITLRILFRPVASQLPRIFTSIGEDYDERVLPSITTEILKSVVARFDA GELITQRELVSRQVSDDLTERAATFGLILDDVSLTHLTFGKEFTEAVEAKQVAQQEAERARFVVEKAEQQKKAAIISAEGDSKAAELIANSLATAGDGLIELRKLEAAEDIAYQLSRSRNITYLPAGQSVLLQLPQ.

[0007] The recombinant human PHB1 protein was prepared using a prokaryotic expression system with pET-28a(+) as the expression vector and Escherichia coli BL21(DE3) as the host bacterium. It was expressed using an N-terminal 6×His tag fusion and purified by nickel ion affinity chromatography (Ni-NTA). The His tag was cleaved using enterokinase.

[0008] In a second aspect, the present invention provides a medicament for treating diabetic cardiomyopathy, comprising the recombinant human PHB1 protein described in the first aspect.

[0009] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0010] Thirdly, the present invention provides the use of the recombinant human PHB1 protein described in the first aspect in the preparation of a medicament for treating diabetic cardiomyopathy.

[0011] Fourthly, the present invention provides the use of the recombinant human PHB1 protein described in the first aspect in the preparation of drugs that inhibit cardiomyocyte apoptosis or reduce myocardial inflammation.

[0012] Recombinant PHB1 protein exerts its cardioprotective effect through the following mechanisms: (1) Improve cardiomyocyte viability: Exogenous recombinant PHB1 protein can directly act on cardiomyocytes and significantly improve the survival rate of cardiomyocytes damaged by high glucose / palmitic acid; (2) Reduce the production of reactive oxygen species (ROS): PHB1 reduces the release of mitochondrial reactive oxygen species (mtROS) and alleviates oxidative stress damage; (3) Inhibit NLRP3 inflammasome activation and apoptosis: PHB1 inhibits NLRP3 inflammasome expression and reduces the synthesis of inflammatory factors such as active TNF-α; at the same time, it reduces the expression of active Caspase-3, that is, reduces myocardial cell apoptosis.

[0013] Beneficial effects: 1) Significant cardioprotective effect: Recombinant PHB1 protein treatment significantly improved cardiomyocyte viability in the DCM model, reduced the level of myocardial inflammatory factor TNF-α, decreased the expression of active Caspase-3 and NLRP3, and alleviated myocardial inflammatory cell infiltration; 2) Reduce oxidative stress: Recombinant PHB1 protein treatment effectively reduces the production of reactive oxygen species (ROS) in cardiomyocytes induced by high glucose / palmitic acid, thus alleviating oxidative stress damage; 3) Good safety: The recombinant protein has the same sequence as the endogenous PHB1 protein and low immunogenicity; the endotoxin content is <0.25 EU / mg, which meets the pharmaceutical standards for injection. 4) Clear dose-response relationship: It exhibits a good dose-response relationship within the dose range of 0.5-2.0 mg / kg; it has a wide therapeutic window and high safety. Attached Figure Description

[0014] Figure 1 Figure showing the results of recombinant PHB1 protein enhancing the viability of high glucose / palmitate-induced cardiomyocytes; Figure 2 The results of reducing high glucose / palmitate-induced reactive oxygen species (ROS) production in cardiomyocytes by recombinant PHB1 protein (DCFH-DA fluorescence staining). Figure 3 In Western blot experiments, recombinant PHB1 protein reduced NLRP3 inflammasome expression and apoptosis-related Caspase-3 expression in high glucose / palmitate induced cardiomyocytes. Figure 4 In the immunoblotting experiment, recombinant PHB1 protein reduced the expression of NLRP3 inflammasome and apoptosis-related Caspase-3 in cardiac cells of diabetic cardiomyopathy mice; Figure 5 To reduce serum TNF-α levels in diabetic cardiomyopathy mice by recombinant PHB1 protein. Detailed Implementation

[0015] The present invention will be described in detail below with reference to specific embodiments: Example 1: Preparation of recombinant human PHB1 protein 1.1 Construction of recombinant expression vectors: 1) Vector selection: pET-28a(+) prokaryotic expression vector was selected, which contains a 6×His tag at the N-terminus and a thrombin cleavage site (LVPRGS). After cleavage, the N-terminus of the PHB1 protein has two additional amino acids, Gly-Ser. The primers were designed to include enterokinase cleavage sites, and the His tag was removed to obtain the natural PHB1 protein.

[0016] 2) Primer design and synthesis Based on the human PHB1 coding region sequence, specific primers were designed to introduce restriction enzyme sites: PHB1-F (SEQ ID NO.2): 5'-CATATGGACGACGACGACAAGGCTGCCAA-3'; PHB1-R (SEQ ID NO.3): 5'-CCGCTCGAGTCACTGGGGCAGCTGGAGGACACGGACTC-3'; The upstream primer introduced the Nde I site (CATATG, containing the start codon ATG) and the enterokinase cleavage site (ENLYFQGS), and the downstream primer introduced the Xho I site (CTCGAG). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and purified by PAGE.

[0017] 3) Target gene amplification: The human PHB1 coding region was amplified by PCR. The amplification system was 10×PCR Buffer (containing Mg). 2 + 5 μL of dNTP Mix (10 mM each), 1 μL of upstream primer PHB1-F (10 μM), 1 μL of downstream primer PHB1-R (10 μM), 1 μL of template (human heart cDNA library, 50 ng / μL), 0.5 μL of high-fidelity DNA polymerase (Phusion, 2 U / μL), and 40.5 μL of sterile double-distilled water. The amplification program was: 98 °C for 30 s; 98 °C for 10 s, 58 °C for 30 s, 72 °C for 30 s, 35 cycles; 72 °C for 5 min.

[0018] 4) Enzyme digestion and ligation Double enzyme digestion reaction: The system consisted of 2 μg of PCR product or pET-28a vector, 3.1 5 μL of 10×NEBuffer, 1 μL of NdeI (10 U / μL), 1 μL of Xho I (10 U / μL), and sterile double-distilled water to a final volume of 50 μL; the digestion was carried out in a water bath at 37℃ for 3 h; the digested products were separated by 1% agarose gel electrophoresis and recovered using a DNA gel recovery kit (Tiangen Biotech, DP209); Ligation reaction: The system consisted of 50 ng of digested pET-28a vector, 150 ng of digested PHB1 fragment, 1 μL of 10×T4 DNA ligase buffer, 0.5 μL of T4 DNA ligase (350 U / μL), and sterile double-distilled water to a final volume of 10 μL; ligation was carried out overnight at 16°C (12-16 h).

[0019] 5) Transformation and identification of recombinant plasmids Transformation of E. coli DH5α competent cells: ① Take 5 μL of the ligation product and add it to 50 μL of DH5α competent cells (Tiangen Biotech, CB101), and incubate on ice for 30 min; ② Heat shock at 42℃ for 90 seconds, then immediately ice bath for 2 minutes; ③ Add 500 μL of sterile LB medium and revive at 37℃ and 180 rpm for 1 h; ④ Spread 200 μL of bacterial suspension onto an LB agar plate containing 50 μg / mL kanamycin; ⑤ Incubate at 37℃ for 12-16 h.

[0020] Screening and identification of positive clones: ① Pick a single colony and verify it by colony PCR (primers are PHB1-F and PHB1-R). ② Plasmids were extracted from positive clones and verified by Nde I / Xho I double digestion (expected band: vector 5329 bp + insert fragment 837 bp); ③ Sequencing verification: Sanger sequencing was commissioned to Sangon Biotech, using T7 universal sequencing primers.

[0021] 1.2 Transformation and Induced Expression 1) Transformation of the expression strain: The recombinant plasmid pET-28a-PHB1 with correct sequencing was transformed into the expression host strain Escherichia coli BL21(DE3) (Cytiva, 27-1542-01).

[0022] 2) Optimization of induction expression conditions Seed culture preparation: Take the inoculum from the -80℃ freezer and streak it on LB agar plates containing kanamycin (50 μg / mL). Incubate at 37℃ for 12 h. Pick a single colony and inoculate it into 5 mL of LB medium (containing 100 μg / mL ampicillin). Incubate overnight (12 h) at 37℃ with shaking at 220 rpm. Scale-up culture and induction: Inoculate 200 mL of LB medium (containing 50 μg / mL kanamycin) at a 1:100 ratio, and culture at 37℃ with shaking at 220 rpm. OD is measured every 30 min. 600 , to OD 600 = 0.6-0.8 (logarithmic growth phase), add isopropyl-β-D-thiogalactoside (IPTG) for induction, the final concentration of the inducer is 0.5 mM; 25℃ (enterokinase cleavage requires maintaining protein activity, the induction temperature is optimized), induction time is 12-16 h, and the rotation speed is 180 rpm.

[0023] 3) Bacterial cell collection and lysis Collect bacterial cells: After induction, collect bacterial cells by centrifugation at 4℃ and 8000 rpm for 10 min. Wash the bacterial cells twice with pre-cooled lysis buffer, weigh the bacterial cell pellet, and resuspend it at a ratio of 1 g bacterial cells to 10 mL lysis buffer. Lysis buffer formulation: NaH2PO4 50 mM (pH 8.0), NaCl 300 mM, imidazole 10 mM (to reduce nonspecific binding), glycerol 10% (v / v), benzyl sulfonyl fluoride (PMSF) 1 mM (added fresh), lysozyme 1 mg / mL (added fresh), DNase I 10 μg / mL (added fresh to reduce viscosity), β-mercaptoethanol 5 mM (added fresh to protect the protein); Lysis conditions: ice bath sonication power 300 W, working for 3 s / intermittent for 5 s, total time 15 min; lysis buffer centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant (containing soluble His-PHB1 fusion protein) was collected.

[0024] 1.3 Purification and Endotoxin Removal: 1) Nickel ion affinity chromatography (Ni-NTA) Purification chromatography column: Ni-NTA Agarose (Qiagen, 30210) or Chelating Sepharose FastFlow (Cytiva, 17057501), column volume 5 mL; purification steps are shown in Table 1 below: Table 1 Purification Steps , Imidazole competitively binds to Ni 2+The His-tagged fusion protein was eluted; the purity of the eluent was immediately determined by SDS-PAGE.

[0025] 2) Tag Removal: The His tag is removed, and enterokinase cleavage is performed. The steps are shown in Table 2 below: Table 2 Label Removal Procedure , After cutting, the His tag and enterokinase were removed by nickel ion affinity chromatography (enterokinase has no His tag, and the flow-through contains the target PHB1 protein; the His tag is bound to the Ni-NTA column), and the flow-through (containing native PHB1 protein) was collected.

[0026] 3) Gel filtration chromatography (fine purification) Chromatography column: Superdex 200 Increase 10 / 300 GL (Cytiva, 28990944); Operating conditions: Equilibration buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol), flow rate: 0.5 mL / min, loading volume: 500 μL (protein concentration ≤10 mg / mL); detection wavelength: 280 nm; Natural PHB1 with its His tag removed is either a monomer (~30 kDa) or a dimer (~60 kDa), and elution peaks within the corresponding molecular weight range are collected.

[0027] 4) Endotoxin removal: Endotoxins were removed using a combination of Triton X-114 phase separation and anion exchange chromatography. The operational steps are shown in Tables 3 and 4. Table 3. Operating Procedures for the Triton X-114 Phase Separation Method , Triton X-114 forms a cloud point at 37°C, and the lipophilic endotoxin partitions into the detergent phase.

[0028] Table 4. Operating Procedures for Anion Exchange Chromatography , Chromatography column: Q Sepharose Fast Flow (Cytiva, 17051001); At pH 7.5, endotoxin (pI < 2.0) binds to the Q column, while PHB1 (pI 5.57) does not bind and flows through.

[0029] 5) Ultrafiltration concentration and buffer replacement: Add the purified protein solution to an ultrafiltration tube (10 kDa molecular weight cutoff), centrifuge at 4℃ and 4000 rpm for concentration; dilute and concentrate repeatedly 3 times with preparation buffer (20 mM phosphate buffer, pH 7.4, 150 mM NaCl, 5% mannitol); adjust the final concentration to 2-5 mg / mL.

[0030] Protein identification 1) SDS-PAGE electrophoresis analysis: The theoretical molecular weight of the His-PHB1 fusion protein is approximately 33.8 kDa (His tag approximately 4 kDa + PHB1 29.8 kDa); the molecular weight of the PHB1 protein after enterokinase cleavage is approximately 29.8 kDa; purity ≥95%.

[0031] 2) Western blot identification Antibody selection: His-PHB1 fusion protein: anti-His tag antibody (mouse, Proteintech, 66005-1-Ig) 1:2000; HRP-anti-His antibody (mouse, Proteintech, HRP-66005) 1:5000; PHB1 protein: anti-PHB1 antibody (rabbit, Proteintech, 10761-1-AP) 1:1000; HRP-labeled goat anti-rabbit IgG secondary antibody (Proteintech, SA00001-2) 1:5000.

[0032] 3) Endotoxin detection (LAL gel method) reagents: Limulus amebocyte lysate (TAL, Fuzhou Xinbei Biochemical Industry Co., Ltd., sensitivity 0.25 EU / mL); Procedure (gel method): Take 10 μL of protein sample (dilute with water to the appropriate concentration for LAL test); add 0.1 mL of Limulus amebocyte lysate (LAL) reagent and mix gently; incubate in a water bath at 37℃ for 60 ± 2 min, then remove and observe; Result determination: Negative: When the test tube is tilted at 45°, the contents are a firm gel that does not flow; Positive: The contents are fluid or semi-gelatinous; The endotoxin content in the sample was <0.25 EU / mg protein (meets the standards for injectable drugs).

[0033] 4) The protein concentration determination and total yield results are shown in Table 5: Table 5 Protein Concentration Determination and Overall Yield

[0034] Example 2: Protective effect of recombinant PHB1 protein against high glucose / palmitate-induced cardiomyocyte injury (in vitro experiment) Cell culture: H9C2 rat cardiomyocytes were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and incubated at 37℃ in a 5% CO2 incubator. Subsequent experiments were carried out when the cell confluence reached 80%-90%.

[0035] Cell model establishment: H9C2 cells were treated with high glucose (30 mM glucose) and palmitic acid (200 μM) for 48 h to establish an in vitro diabetic cardiomyocyte injury model.

[0036] Experimental Groups: Vehicle group: High glucose / palmitic acid treatment + PBS control rPHB1 group: high glucose / palmitic acid treatment + recombinant PHB1 protein (natural PHB1, His tag removed, 10 μg / mL) Test indicators and results: (1) Cell viability assay (CCK-8 assay): such as Figure 1 As shown, compared with the Vehicle group (relative viability set at 1.0), the relative viability of cardiomyocytes in the rPHB1 group was significantly increased (approximately 1.5-fold, p<0.001), indicating that recombinant PHB1 protein can significantly improve high glucose / palmitic acid-induced cardiomyocyte damage and increase cell survival rate; (2) Detection of reactive oxygen species (ROS) (DCFH-DA fluorescence staining): such as Figure 2 As shown, the Vehicle group showed obvious green fluorescence (ROS positive signal), while the fluorescence signal of the rPHB1 group was significantly weakened, indicating that recombinant PHB1 protein can effectively reduce the production of reactive oxygen species in cardiomyocytes induced by high glucose / palmitic acid and alleviate oxidative stress damage. (3) NLRP3 inflammasome and apoptosis detection (Western blot): such as Figure 3 As shown, compared with the Vehicle group, the rPHB1 group showed significantly reduced NLRP3 protein expression and significantly reduced Cleaved Caspase-3 (activated Caspase-3) expression, while the total Caspase-3 level did not change significantly. This indicates that recombinant PHB1 protein can inhibit NLRP3 inflammasome activation and reduce cardiomyocyte apoptosis.

[0037] Example 3: Establishment of a mouse model of diabetic cardiomyopathy, treatment with recombinant PHB1 protein, and detection of myocardial tissue (in vivo experiment) 1) Experimental animals: 6-week-old male C57BL / 6J mice, weighing 18-22 g, were used for experiments after 1 week of acclimatization feeding.

[0038] 2) Establishment of DCM mouse model and drug administration regimen: such as Figure 4 As shown above, the experimental procedure is as follows: Days 1-5: Model group mice were intraperitoneally injected with streptozotocin (STZ, 50 mg / kg / d) for 5 consecutive days; blank control group mice were intraperitoneally injected with an equal volume of physiological saline. Day 42: Measure fasting blood glucose to confirm successful establishment of the diabetes model (fasting blood glucose ≥11 mmol / L). Starting from Day 43: Recombinant PHB1 protein treatment was initiated. Mice in the treatment group were injected intraperitoneally with recombinant PHB1 protein (natural PHB1, His tag removed, 2 mg / kg) twice a week for 4 weeks; the model control group was injected intraperitoneally with an equal volume of physiological saline.

[0039] 3) Determining the effective therapeutic dose: Low-dose group (0.5 mg / kg): showed some cardioprotective effect, with a decrease of approximately 20% in NLRP3 and active Caspase-3 expression; The medium-dose group (1.0 mg / kg) showed significant cardioprotective effects, with a reduction of approximately 45% in NLRP3 and active Caspase-3 expression; High-dose group (2.0 mg / kg): showed therapeutic effects comparable to those of the medium-dose group, but no significant dose-dependent enhancement effect was observed; Based on the conversion factor between mouse and human body surface area (approximately 1:12), the estimated effective therapeutic dose for humans is approximately 0.04-0.17 mg / kg body weight, preferably 0.08 mg / kg body weight. For a 60 kg adult, the single dose is approximately 2.4-10 mg, preferably 5 mg.

[0040] The administration frequency is once every 3 days for 4-8 weeks, or adjusted according to the patient's condition.

[0041] 4) Indicator testing and results: (1) Detection of NLRP3 inflammasomes and apoptosis in myocardial tissue (Western blot): such as Figure 4 As shown below, compared with the Vehicle group, the rPHB1 treatment group showed significantly decreased NLRP3 protein expression and significantly reduced Cleaved Caspase-3 expression in the myocardial tissue of mice, while the total Caspase-3 level remained unchanged. These results are consistent with in vitro cell experiments (…). Figure 3 The results were consistent, indicating that recombinant PHB1 protein can effectively inhibit NLRP3 inflammasome activation and reduce cardiomyocyte apoptosis both in vivo and in vitro. (2) Serum inflammatory factor detection (ELISA): such as Figure 5As shown, compared with the Vehicle group, the serum TNF-α level of mice in the rPHB1 group was significantly reduced (from about 1500 pg / mL to about 600 pg / mL), indicating that recombinant PHB1 protein can significantly inhibit the systemic inflammatory response in DCM mice.

[0042] The experimental results showed that, compared with the model control group, the expression of myocardial NLRP3 and Cleaved Caspase-3 and the serum TNF-α level in the treatment group mice were significantly reduced (all P < 0.01), proving that recombinant PHB1 protein (with His tag removed) can effectively treat diabetic cardiomyopathy.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant human PHB1 protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A drug for treating diabetic cardiomyopathy, characterized in that: It contains the recombinant human PHB1 protein as described in claim 1.

3. The drug according to claim 2, characterized in that: It also includes pharmaceutically acceptable carriers.

4. The use of the recombinant human PHB1 protein according to claim 1 in the preparation of a medicament for treating diabetic cardiomyopathy.

5. The use of the recombinant human PHB1 protein according to claim 1 in the preparation of a drug for inhibiting cardiomyocyte apoptosis caused by diabetes or reducing myocardial inflammation.

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