Use of a PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis

Abstract

The application relates to the field of biological medicine, and discloses an application of knocking down a PDK1 gene in inhibiting hypoxia-induced renal fibrosis, wherein the recombinant lentivirus composition is made of a recombinant lentivirus plasmid carrying a PDK1 gene sequence, a packaging auxiliary plasmid, an envelope protein expression plasmid and polybrene. The composition targets and knocks down the expression of the PDK1 gene in renal interstitial fibroblasts, removes the phosphorylation inhibition on pyruvate dehydrogenase, promotes pyruvate to enter the tricarboxylic acid cycle, and blocks the aerobic glycolysis metabolic reprogramming of cells. The scheme cuts off the energy substrate supply for abnormal cell proliferation and activation, inhibits the TGF-beta 1 / Smad fibrosis-promoting signal path, reduces the extracellular matrix deposition, induces pathological cells to start the apoptosis program, and inhibits tissue fibrosis from the metabolic level.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of knocking down the PDK1 gene in inhibiting hypoxia-induced renal fibrosis. Background Technology

[0002] Renal fibrosis is a common pathological pathway in the end-stage of various chronic kidney diseases. Its histological characteristics include abnormal proliferation and phenotypic transformation of renal interstitial fibroblasts, as well as extensive deposition of extracellular matrix in the interstitial spaces. During disease progression, damage to local microvessels and reduced blood perfusion lead to a chronic hypoxic state in the renal tissue. This hypoxic microenvironment is the essential underlying condition for triggering fibroblast activation and driving the fibrotic process.

[0003] Current interventions for renal fibrosis primarily focus on controlling inflammatory responses or blocking pro-fibrotic signaling pathways. In practice, researchers use anti-inflammatory drugs to reduce early tissue damage or develop specific inhibitors to intervene in transforming growth factor and its downstream signaling pathways, thereby reducing the transcriptional levels of matrix proteins. In addition, conventional approaches also include the use of vasodilators to improve local blood circulation or the use of specific enzyme modulators to intervene in the already formed extracellular matrix network structure.

[0004] Current technologies have failed to block the abnormal energy metabolism reprogramming process of fibroblasts in hypoxic microenvironments. Under hypoxic stimulation, fibroblasts alter their basal energy metabolism pattern, shifting their energy acquisition pathway to aerobic glycolysis, thereby providing sufficient energy substrates and metabolic intermediates for excessive cell proliferation and continuous activation. Existing interventions act on downstream execution processes such as signal transduction, failing to cut off the material and energy supply basis for pathogenic cells to maintain abnormal activities at the metabolic root, making it difficult to specifically inhibit the pathological process of fibrosis with the support of metabolic substrates. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an application of PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a recombinant lentiviral composition for inhibiting hypoxia-induced renal fibrosis, employing the following technical solution: A recombinant lentiviral composition for inhibiting hypoxia-induced renal fibrosis is made from the following raw materials in parts by weight: 3-5 parts of a recombinant lentiviral plasmid carrying a PDK1 gene sequence; 2-4 parts of a packaging helper plasmid; 1-2 parts of an envelope protein expression plasmid; and 0.4-1.0 parts of polybrene.

[0007] By employing the above-mentioned technical solution, and utilizing a recombinant lentiviral plasmid co-packaging system targeting the PDK1 gene sequence and polybrene, the pathological progression of renal fibrosis is inhibited. The specific intervention mechanism includes the following steps: The first step is entry into the host cell: the polybrene in the composition neutralizes the electrostatic repulsion between the surface of the virus particles and the cell membrane, thereby increasing the efficiency of lentiviruses in penetrating the cell membrane and entering renal interstitial fibroblasts.

[0008] The second step is gene-targeted silencing: the target sequence carried by the recombinant lentivirus is transcribed into short hairpin RNA in the host cell nucleus. After being processed by endogenous enzymes, it recognizes and degrades the mRNA transcribed from the PDK1 gene through base complementarity pairing, thereby blocking the translation of the PDK1 protein.

[0009] The third step is to block the glycolysis pathway: the expression level of PDK1 protein decreases, which relieves the inhibition of phosphorylation of pyruvate dehydrogenase, promotes the re-entry of pyruvate into the mitochondrial tricarboxylic acid cycle, and blocks the aerobic glycolysis pathway that allows cells to obtain abnormal proliferative energy.

[0010] The fourth step is to inhibit matrix deposition: After the abnormal glycolysis pathway is blocked, the cells lack energy substrates and metabolic intermediates, and cannot maintain the cascade transcriptional activity of the TGF-β1 / Smad pro-fibrosis signaling pathway, thus reducing the synthesis and deposition of extracellular matrix components such as type II collagen, fibronectin and connective tissue growth factor.

[0011] The fifth step is to reverse the anti-apoptotic phenotype: changes in energy metabolism homeostasis lead to changes in mitochondrial membrane permeability, reduce the ratio of Bcl-2 to Bax expression, activate the Caspase 9 and Caspase 3 cascade, and induce pathologically activated renal interstitial fibroblasts to initiate apoptosis.

[0012] Preferably, the mass ratio of the recombinant lentiviral plasmid, packaging helper plasmid, and envelope protein expression plasmid is 4:3:1.5; the PDK1 gene targeting sequence carried in the recombinant lentiviral plasmid is any one of the first target sequence, the second target sequence, or the third target sequence.

[0013] By adopting the above technical solution, the core plasmid ratio required for virus packaging is limited, ensuring the biosynthetic efficiency and product stability of the virus particle assembly process, and providing sequence types with clear targeted cleavage efficiency.

[0014] Preferably, the raw materials also include an additional screening component, wherein the additional screening component is puromycin, and the dosage is 0.1 to 0.2 parts.

[0015] By adopting the above technical solution, drug screening pressure is provided for transfected host cells, background cells that have not been successfully infected with lentiviruses are eliminated, and a uniform gene knockdown cell population is obtained.

[0016] Preferably, the packaging helper plasmid and the envelope protein expression plasmid are composed of a first packaging plasmid component carrying the gag / pol gene, a second packaging plasmid component carrying the rev gene, and an envelope expression plasmid component carrying the VSV-G gene.

[0017] By adopting the above technical solution, the genetic backbone of the lentiviral packaging system is disassembled, eliminating the physiological risk of genome recombination producing wild-type viruses with replication capabilities and improving the biosafety of the composition.

[0018] Preferably, the recombinant lentiviral plasmid is prepared by linearizing a lentiviral backbone plasmid carrying an resistance expression cassette through double restriction enzyme digestion, and then ligating it with an annealed shRNA oligonucleotide double strand targeting the PDK1 gene at 14-22°C for 1-16 hours.

[0019] By adopting the above technical solution, the molecular cloning process parameters of the core active ingredient are clarified, ensuring the connection efficiency and sequence orientation accuracy of the oligonucleotide targeting double strand with the viral backbone vector.

[0020] Secondly, the present invention provides a method for preparing a recombinant lentiviral composition for inhibiting hypoxia-induced renal fibrosis, employing the following technical solution: A method for preparing a recombinant lentiviral composition for inhibiting hypoxia-induced renal fibrosis includes the following steps: S100, the recombinant lentiviral plasmid, packaging helper plasmid and envelope protein expression plasmid are mixed in proportion, and after adding transfection reagent, they are co-transfected into host packaging cells; S200 involves continuously culturing transfected host packaging cells in a medium containing fetal bovine serum and collecting the cell culture supernatant containing viral particles. S300: The collected cell culture supernatant is centrifuged and filtered to remove cell debris, and then polybrene in the specified ratio is added and mixed to obtain the recombinant lentivirus composition.

[0021] By adopting the above technical solution, the virus particle packaging is completed using the biological translation and assembly system of the host cell, and the final product is determined by combining centrifugation filtration and chemical reagent compounding processes, thereby obtaining an intervention composition formulation that can be directly applied.

[0022] Preferably, in step S100, the host packaging cells are pretreated as follows: the revived 293T cells are passaged to the 3rd to 5th generation, and when the cells grow to the logarithmic growth phase and the cell confluence reaches 70% to 80%, they are washed with phosphate buffer free of calcium and magnesium ions, and then used for subsequent plasmid co-transfection.

[0023] By adopting the above technical solutions, the physiological metabolic state of the host cell is regulated to the active division phase, thereby improving the efficiency of transmembrane transport of exogenous plasmid nucleic acids and their conversion into expression in the cell nucleus.

[0024] Preferably, the specific process parameters for step S200 are as follows: the transfected cells are placed in a constant temperature incubator at 37°C and 5% carbon dioxide by volume, and the cell culture supernatant is collected twice, at 48 hours and 72 hours after transfection, and the two collections are combined.

[0025] By adopting the above technical solution, the main peak time period from intracellular assembly to secretion and release of lentiviral particles is covered, thereby increasing the total amount of virus produced per unit volume of cell culture system.

[0026] Preferably, in step S300, the performance parameters of the obtained recombinant lentiviral composition are controlled within the range of: viral titer of 1E+8 TU / mL to 6E+8 TU / mL.

[0027] By adopting the above technical solutions, a quantitative potency control standard for the final product of the composition is established, ensuring the accuracy of dosage control and the reproducibility of gene knockdown effects in cell and in vivo intervention experiments.

[0028] Thirdly, this invention provides an application of PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis, employing the following technical solution: An application of PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis, wherein the drug uses the recombinant lentiviral composition as the active ingredient to target and knock down the expression level of the PDK1 gene in renal interstitial fibroblasts; the inhibition of hypoxia-induced renal fibrosis specifically includes: blocking the aerobic glycolytic metabolic pathway of renal interstitial fibroblasts; inhibiting the hypoxia-induced TGF-β1 / Smad pro-fibrotic signaling pathway, reducing the synthesis and deposition of extracellular matrix-related proteins; reversing the anti-apoptotic properties of hypoxic cells, and inducing apoptosis in pathologically activated renal interstitial fibroblasts.

[0029] By adopting the above technical solutions, effective active agents with specific target intervention effects are provided for the pathological turning point of metabolic reprogramming caused by hypoxic microenvironment. These agents intervene in the process of renal fibrosis from three dimensions: blocking abnormal metabolism, weakening signal transduction, and initiating cell apoptosis, and establish corresponding medical drug preparation pathways.

[0030] This invention provides an application of PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis. It has the following beneficial effects: 1. This invention constructs a recombinant viral vector carrying a specific sequence to target and knock down the expression of the kinase PDK1 gene in renal interstitial fibroblasts. This relieves the inhibition of PDK1 phosphorylation of pyruvate dehydrogenase (PDH), prompting pyruvate to enter the tricarboxylic acid cycle and blocking hypoxia-induced aerobic glycolysis metabolic reprogramming. This metabolic intervention cuts off the energy substrate supply required for cell proliferation and activation, overcoming the limitations of traditional interventions that only act on downstream signal transduction, thus depriving the pathological process of fibrosis of its material support and achieving specific inhibition of the progression of renal fibrosis.

[0031] 2. This invention weakens the cascade activation effect of transforming growth factor and its downstream pro-fibrotic signaling pathways by blocking abnormal glycolytic metabolic pathways. In the absence of metabolic substrates, pathologically ill fibroblasts lose their ability to maintain high levels of transcriptional activity, reducing the synthesis rate of extracellular matrix components such as collagen and fibronectin. This mechanism intervenes in the fibrotic stage, reducing abnormal deposition of matrix components in the renal interstitium and preventing tissue fibrosis at the solid level.

[0032] 3. This invention alters the energy metabolism homeostasis of hypoxic cells, inducing changes in mitochondrial membrane permeability in abnormally activated cells, downregulating the expression ratio of anti-apoptotic and pro-apoptotic proteins, and activating the biochemical cascade of downstream apoptotic execution proteases. This reaction reverses the hypoxic microenvironment, endowing renal interstitial fibroblasts with an anti-apoptotic pathological phenotype, inducing pathologically activated effector cells to initiate apoptosis, creating conditions for the body to clear abnormally proliferating cells and promoting tissue repair. Attached Figure Description

[0033] Figure 1 The figure shows the detection results of the knockdown efficiency of the PDK1 gene by the recombinant lentiviral targeting sequence. Figure 2 Figure 1 shows the detection results of physicochemical indicators of cellular glucose metabolism and expression levels of key enzyme genes in each group. Figure 3 The graph shows the results of detecting the expression levels of gene markers for cell proliferation and activation in each group. Figure 4 Figure 1 shows the results of detecting the expression levels of profibrotic pathways and extracellular matrix-related genes and proteins in each group of cells. Figure 5 The graph shows the detection results of cell apoptosis rate and expression levels of apoptosis-related genes in each group. Figure 6 Figure showing the results of differential analysis and pathway enrichment of cell transcriptome sequencing data; Figure 7This figure shows a comparison between the sequencing results of differentially expressed genes and the verification results of quantitative polymerase chain reaction. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Please see the appendix Figure 1 - Appendix Figure 7 This invention provides an application of PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis. The main raw materials and reagents used in the following examples and comparative examples are as follows. Unless otherwise specified, all reagents are commercially available analytical grade or higher products.

[0036] The primary yak kidney interstitial fibroblasts used in this invention were extracted from healthy yak tissue and obtained by primary culture and passage.

[0037] The recombinant lentiviruses used to construct the PDK1 gene knockdown model were obtained by synthesizing shRNA sequences that specifically target different sites of the PDK1 gene and assembling them using a lentivirus packaging system. These corresponded to the sh27 group, sh28 group, and sh29 group, which had different knockdown efficiencies in the test, respectively, as the intervention vectors for which sh29 was the most preferred.

[0038] The PDK1 gene overexpression vector used to construct the reverse validation model was obtained by cloning the full-length coding sequence of the yak PDK1 gene into a conventional lentiviral expression vector.

[0039] The resistance screening reagent used in the experiment was puromycin, whose chemical name is 3'-(α-amino-p-methoxyhydrocinnamamido)-3'-deoxy-N,N-dimethyladenosine, CAS number is 58-58-2, and molecular formula is C22H29N7O5.

[0040] Polybrene, used to assist in lentiviral transfection, has CAS number 28728-55-4.

[0041] Preparation Example 1: This preparation example provides a recombinant lentivirus targeting the PDK1 gene, comprising the following steps: Based on the first specific target of the PDK1 gene, the corresponding shRNA sequence was synthesized, and the shRNA sequence was assembled into lentivirus particles using a conventional lentivirus packaging system. The corresponding lentivirus transfection solution was collected and used as the sh27 group vector for knockdown intervention in subsequent experiments.

[0042] Preparation Example 2: This preparation example provides a recombinant lentivirus targeting the PDK1 gene, comprising the following steps: Based on the second specific target of the PDK1 gene, the corresponding shRNA sequence was synthesized, and the shRNA sequence was assembled into lentivirus particles using a conventional lentivirus packaging system. The corresponding lentivirus transfection solution was collected and used as the sh28 group vector for knockdown intervention in subsequent experiments.

[0043] Preparation Example 3: This preparation example provides a recombinant lentivirus targeting the PDK1 gene, comprising the following steps: Based on the third specific target of the PDK1 gene, the corresponding shRNA sequence was synthesized. The shRNA sequence was then assembled into lentiviral particles using a conventional lentiviral packaging system. The corresponding lentiviral transfection solution was collected, and its viral titer was measured to be 6E+8TU / mL. This preparation example represents the optimal group for targeted knockdown efficiency and will serve as the sh29 group vector for subsequent knockdown interventions.

[0044] Example 1: This embodiment provides a method for constructing a yak kidney interstitial fibroblast cell line with PDK1 gene knockdown, including the following steps: S100, well-grown fourth-generation yak kidney interstitial fibroblasts were prepared into a cell suspension, and then... The samples were seeded at a density of 10% fetal bovine serum into 24-well plates, and DMEM complete medium containing 10% fetal bovine serum was added. The plates were then incubated at 37°C in a 5% CO2 incubator. S200, when the cell confluence reaches 60%, add the lentivirus transfection solution for targeted knockdown of the PDK1 gene constructed in Preparation Example 3 at a ratio of 30 MOI; After culturing in S300 for 24 hours, the medium was replaced with fresh DMEM complete medium. Once the cells reached confluence, medium containing 1.0 μg / mL puromycin was added for three consecutive generations of resistance selection. The selected cells were collected to obtain a recombinant cell model with stable knockdown of PDK1 gene expression.

[0045] Example 2: This embodiment provides a method for constructing a yak kidney interstitial fibroblast cell line with PDK1 gene knockdown, including the following steps: S100, well-grown fourth-generation yak kidney interstitial fibroblasts were prepared into a cell suspension, and then... The samples were seeded at a density of 10% fetal bovine serum into 24-well plates, and DMEM complete medium containing 10% fetal bovine serum was added. The plates were then incubated at 37°C in a 5% CO2 incubator. S200, when the cell confluence reaches 70%, add the lentivirus transfection solution for targeted knockdown of the PDK1 gene constructed in Preparation Example 3 at a ratio of 50 MOI. After culturing in S300 for 24 hours, the medium was replaced with fresh DMEM complete medium. Once the cells reached confluence, medium containing 1.5 μg / mL puromycin was added for three consecutive generations of resistance selection. The selected cells were collected to obtain a recombinant cell model with stable knockdown of PDK1 gene expression.

[0046] Example 3: This embodiment provides a method for constructing a yak kidney interstitial fibroblast cell line with PDK1 gene knockdown, including the following steps: S100, well-grown fourth-generation yak kidney interstitial fibroblasts were prepared into a cell suspension, and then... The samples were seeded at a density of 10% fetal bovine serum into 24-well plates, and DMEM complete medium containing 10% fetal bovine serum was added. The plates were then incubated at 37°C in a 5% CO2 incubator. S200, when the cell confluence reaches 80%, add the lentivirus transfection solution for targeted knockdown of the PDK1 gene constructed in Preparation Example 3 at a ratio of 70 MOI; After culturing in S300 for 24 hours, the medium was replaced with fresh DMEM complete medium. Once the cells reached confluence, medium containing 2.0 μg / mL puromycin was added for three consecutive generations of resistance selection. The selected cells were collected to obtain a recombinant cell model with stable knockdown of PDK1 gene expression.

[0047] Comparative Example 1: Compared with Example 2, the difference is that an empty vector lentivirus transfection solution without carrying a specific PDK1 gene-targeting shRNA sequence was used for transfection, otherwise the same.

[0048] Comparative Example 2: Compared with Example 2, the difference is that a recombinant lentivirus transfection solution carrying the full-length coding sequence of the PDK1 gene was used for transfection to achieve overexpression of the PDK1 gene; all other aspects are the same.

[0049] Comparative Example 3: Compared with Example 2, the difference is that the transfection and resistance screening steps of recombinant lentivirus were not performed. Instead, dichloroacetate, a small molecule glycolysis inhibitor, was directly added to the culture medium during the hypoxia model treatment. All other aspects are the same.

[0050] Comparative Example 4: Compared with Example 2, the difference is that the lentiviral vector carries a knockout sequence that specifically targets the upstream transcription factor HIF-1α gene, rather than the PDK1 gene sequence; all other aspects are the same.

[0051] Comparative Example 5: Compared with Example 2, the difference is that the recombinant lentivirus system and resistance screening process were not used. Instead, the small interfering RNA transient transfection technology was used to introduce the sequence targeting the PDK1 gene into the cells. All other aspects are the same.

[0052] Test Example 1: Gene knockdown efficiency test and optimization of recombinant lentiviral raw materials To verify the effectiveness of the custom raw materials provided in the preparation examples and to screen for the optimal viral vector for constructing cell models in subsequent examples, this test example performed targeted knockdown efficiency testing on the recombinant lentiviruses prepared in Preparation Examples 1 to 3, including the following steps: Recombinant lentiviral transfection solutions prepared in Examples 1, 2, and 3, as well as empty vector lentiviral transfection solution without the target sequence (as a baseline control), were used to independently infect wild-type yak kidney mesenchymal fibroblasts with the same infection parameters, followed by resistance selection. Guanidine isothiocyanate-phenol extract was added to the cell pellets collected from each group to lyse the cells and release nucleic acids. After adding chloroform and shaking, the cells were centrifuged at 12,000 rpm at 4°C. The upper aqueous phase was used to precipitate RNA with isopropanol, washed, dried, and dissolved in deionized water to determine RNA concentration and purity. Using the extracted total RNA as a template, random hexamer primers, deoxyribonucleoside triphosphates, and reverse transcriptase were added to prepare the reaction system. The system was placed in a PCR instrument and incubated at 42°C for 60 minutes to synthesize cDNA, followed by inactivation at 85°C for 5 minutes. Subsequently, an amplification reaction system containing SYBR Green premix, PDK1 gene-specific primers, and cDNA template was prepared, and a parallel control group with β-actin gene as an internal control was set up. The real-time quantitative PCR instrument was programmed with the following steps: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing and extension for 40 seconds, for 40 cycles. Ct values ​​at which fluorescence thresholds were reached were collected, and the relative expression level of PDK1 gene mRNA in each group was calculated using the 2^-ΔΔCt method.

[0053] Table 1. Detection data of relative expression levels of PDK1 gene mRNA by lentiviral raw materials with different target sequences. Test results and mechanism analysis: The custom lentiviral raw material constructed in this technical solution exerts its effect by intervening in the endogenous RNA interference pathway within cells. Its embedded targeting sequence is transcribed into a short hairpin RNA structure within the host cell. Through the base complementarity principle, it specifically recognizes the messenger RNA transcribed from the PDK1 gene, guiding the nuclease complex to target and cleave this mRNA sequence, thus promoting its degradation and blocking the synthesis of related pathogenic proteins at the post-transcriptional level.

[0054] Table 1 shows that, in the baseline control group, the PDK1 gene remained transcribed normally due to the absence of targeted sequence interference, with a mean relative mRNA expression level of 1.02. Preparation Examples 1, 2, and 3 provided targeted intervention materials for different regions of the PDK1 gene sequence. Cells infected with these materials showed a significant decrease in the relative expression level of PDK1 mRNA, confirming that the lentiviral materials provided in the three preparation examples all possess actual gene knockdown function, supporting the overall effectiveness of the specific PDK1-targeting reagent.

[0055] The gradient differences in data among the various preparation examples are determined by the binding matching degree of different nucleotide sequences in the spatial structure of the target gene mRNA. Cells treated with the raw materials from Preparation Example 3 showed a mean relative expression level reduced to 0.35, exhibiting the best target cleavage and silencing efficiency. This test result not only confirmed the effectiveness of the raw materials from the preparation examples but also established Preparation Example 3 as the core intervention vector with the highest knockdown efficiency. This complete gene expression blockade interrupts the abnormal metabolic signal transduction pathways in the hypoxic microenvironment, forming the core basis for the cell models' resistance to aerobic glycolysis and reduction of extracellular matrix deposition in all subsequent examples. Therefore, all subsequent examples uniformly used the optimal raw materials provided by Preparation Example 3 for model construction and comprehensive efficacy comparison testing.

[0056] Test Example 2: Sequencing Identification and Quantitative Validation Test of Biomarkers for Hypoxic Renal Fibrosis Wild-type cells prepared in Comparative Example 1 and stable knockdown cells prepared in Example 2 were cultured in a hypoxic incubator with normoxic and 10% oxygen (v / v) conditions for 48 hours. Cell pellets were collected, and total RNA was extracted. Messenger RNA was enriched using magnetic beads with oligodT, and fragmentation buffer was added to break the nucleic acids. Double-stranded cDNA was synthesized using fragmented RNA as a template via reverse transcription. Transcriptome sequencing libraries were constructed after end repair, A-tailing, and ligation of sequencing adapters. Paired-end sequencing of the libraries was performed using a high-throughput sequencing platform. Adapter sequences and low-quality reads were removed from the raw sequencing data, and the clean sequencing data was aligned to a reference genome to obtain transcript abundance. The logarithmic fold change values ​​of gene expression levels were calculated between the normoxic and hypoxic groups, and between the Example groups and the comparative groups. Differentially expressed genes were screened based on an absolute value of logarithmic fold change greater than 1 and a p-value of less than 0.05 after multiple validation. Differentially expressed genes were mapped to gene ontology libraries and pathway databases. The ATP6V1C2, THBS2, Ppargc1α, and NR4A1 genes were extracted from extracellular matrix receptor interaction, metabolic reprogramming, and acid-base homeostasis pathways. Amplification primers were designed for the ATP6V1C2, THBS2, Ppargc1α, and NR4A1 genes. cDNA was synthesized by reverse transcription using extracted total RNA as a template, and an amplification system containing a fluorescent premix was prepared. Amplification was performed in a real-time quantitative PCR instrument, cycle thresholds were obtained, and the relative expression levels of the ATP6V1C2, THBS2, Ppargc1α, and NR4A1 genes in different groups were calculated.

[0057] Table 2. Sequencing logarithmic fold changes and quantitative PCR relative expression levels of differentially expressed genes. Test Result Analysis: Hypoxic environments alter the energy metabolism patterns and acid-base homeostasis of renal interstitial fibroblasts. Table 2 shows that the ATP6V1C2 gene encodes a vacuolar proton pump subunit. In Comparative Example 1, under hypoxic conditions, the logarithmic folding change of the ATP6V1C2 gene sequencing data was 2.38, and the relative expression level by quantitative PCR was 5.12. In Example 2, under hypoxic conditions, the relative expression level of the ATP6V1C2 gene was lower than that in Hypoxic Comparative Example 1. The test results indicate that knocking down the PDK1 gene blocks the hypoxia-induced increase in acid production.

[0058] The THBS2 gene regulates cell-extracellular matrix interactions. In hypoxia control example 1, the relative expression level of the THBS2 gene by quantitative PCR was 8.04. In hypoxia example 2, the relative expression level of the THBS2 gene was 0.17. The test results indicate that knockdown of the PDK1 gene blocks the synthesis of fibrotic structural proteins.

[0059] The Ppargc1α gene is a transcription factor involved in mitochondrial biosynthesis and oxidative phosphorylation metabolism. In hypoxia control example 1, the relative expression level of the Ppargc1α gene by quantitative PCR was 0.14. In hypoxia example 2, the relative expression level of the Ppargc1α gene was 4.39. The test results indicate that knocking down the PDK1 gene maintains cellular oxidative phosphorylation metabolism.

[0060] The NR4A1 gene is a member of the nuclear receptor family. Data from hypoxia Example 2 showed that the relative expression level of the NR4A1 gene was lower than that in the hypoxia control example 1. The test results indicate that knocking down the PDK1 gene inhibits stress signaling.

[0061] The trends in the expression levels of ATP6V1C2, THBS2, Ppargc1α, and NR4A1 genes in quantitative PCR were consistent with those in transcriptome sequencing. These gene expression changes are correlated with PDK1 gene expression status and cell phenotype, forming a composite of biomarkers for hypoxic renal fibrosis.

[0062] Test Example 3: Comparative Test of Blocking Abnormal Cellular Glycolysis Metabolism Cells from Example 2, Comparative Example 1, and Comparative Example 2 were seeded at the same density in culture dishes and, after adhesion, transferred to an oxygen-deficient incubator with 10% oxygen. Culture supernatant and adherent cells were collected at 12, 48, and 72 hours of hypoxia treatment. Glucose and lactate concentrations in the culture supernatant were measured using a biosensor analyzer, and glucose consumption and lactate production were calculated based on the basal medium concentration. Total RNA was extracted from the adherent cell pellet using lysis buffer, and cDNA was synthesized via reverse transcription. The relative expression levels of Glut1, PKM2, and HK-2 genes were measured using real-time quantitative PCR.

[0063] Test Result Analysis: Hypoxic environments induce metabolic reprogramming in renal interstitial fibroblasts, shifting the cellular energy acquisition pathway from mitochondrial oxidative phosphorylation to aerobic glycolysis. The PDK1 gene regulates the phosphorylation state of pyruvate dehydrogenase, blocking pyruvate from entering the tricarboxylic acid cycle, promoting metabolic shift towards lactate production, and upregulating the expression of enzymes responsible for glucose transport and glycolysis.

[0064] Table 3 shows that prolonged hypoxia increased glucose consumption and lactate production in Comparative Example 1 cells, as well as the relative expression levels of the Glut1, HK-2, and PKM2 genes. The results indicate that wild-type renal interstitial fibroblasts exhibit glycolytic activation under hypoxic conditions.

[0065] In Example 2, during the 72-hour hypoxic period, the highest glucose consumption was 0.76 g / L, the highest lactate production was 0.51 g / L, and the relative expression levels of Glut1, PKM2, and HK-2 genes remained at low levels. All data in Example 2 were lower than those in Comparative Example 1. The test results indicate that knocking down the PDK1 gene disrupts the cascade reaction that leads to aerobic glycolysis, eliminating the energy support for cell proliferation.

[0066] In Comparative Example 2, both the glucose metabolism values ​​and the relative expression levels of key enzymes were higher than in Comparative Example 1. The test results indicate that the PDK1 gene drives hypoxic metabolic reprogramming. Knocking down the PDK1 gene blocks abnormal glycolytic metabolic pathways and has a mechanism of action to intervene in the progression of renal fibrosis.

[0067] Test Example 4: Comparative Test of Cell Proliferation and Activation Inhibition Abilities Cells from Example 2, Comparative Example 1, and Comparative Example 2 were seeded at the same density in multi-well culture plates. After cell adhesion, the culture plates were transferred to an oxygen-deficient incubator with 10% oxygen. Cell pellets were collected after 12, 24, 48, and 72 hours of hypoxia treatment. Total RNA was extracted from the cell pellet using lysis buffer, and cDNA was synthesized via reverse transcription. The relative expression levels of the PCNA and α-SMA genes were determined using real-time quantitative PCR.

[0068] Table 4. Relative expression levels of PCNA and α-SMA genes in cells at different time points under hypoxia. Test Result Analysis: The pathological process of renal fibrosis includes the proliferation of renal interstitial fibroblasts and their transformation into myofibroblast phenotypes. Hypoxic environments regulate cellular energy metabolism pathways, providing the material basis for proliferation and phenotypic transformation. Myofibroblasts express α-SMA and synthesize extracellular matrix.

[0069] Table 4 shows that the relative expression levels of PCNA and α-SMA genes in Comparative Example 1 cells increased with prolonged hypoxia under hypoxic conditions. The results indicate that wild-type renal interstitial fibroblasts initiate proliferation and activation processes under hypoxic stimulation. The relative expression levels of PCNA and α-SMA genes in Comparative Example 2 cells were higher than those in Comparative Example 1 cells. The results suggest that overexpression of the PDK1 gene promotes cell proliferation and activation.

[0070] In Example 2, during the 12-72 hour hypoxia test period, the highest relative expression level of the PCNA gene was 1.12, and the highest relative expression level of the α-SMA gene was 1.17. The data from Example 2 were lower than those from Comparative Example 1. Combined with the changes in glucose metabolism indicators in Example 2, knocking down the PDK1 gene blocked aerobic glycolysis reprogramming, reducing the energy substrate required for cell proliferation and activation. The test results indicate that knocking down the PDK1 gene intervenes in cell phenotypic transformation and inhibits fibroblast proliferation and activation.

[0071] Test Example 5: Comparative Test of Pro-fibrosis Cascade Pathways and Extracellular Matrix Deposition Blockage Cells from Example 2, Comparative Example 1, and Comparative Example 2 were seeded into multi-well culture plates. After cell adhesion, the multi-well culture plates were transferred to an oxygen-deficient incubator with 10% oxygen. Cell pellets were collected after 12, 24, 48, and 72 hours of hypoxia treatment. Total RNA was extracted from the cell pellet using lysis buffer, and cDNA was synthesized by reverse transcription. The relative expression levels of TGF-β1, Smad2, Smad3, Collagen II, FN, and CTGF genes were determined using real-time quantitative PCR.

[0072] Table 5. Relative expression levels of cellular fibrosis-related genes in different groups at different time points under hypoxia. Test Result Analysis: Renal fibrosis is accompanied by activation of pro-fibrotic signaling pathways and extracellular matrix synthesis. Hypoxic environments stimulate renal interstitial fibroblasts to initiate the TGF-β1 / Smad signaling pathway, inducing the transcription of downstream matrix components and increasing the accumulation of type II collagen, fibronectin, and connective tissue growth factor.

[0073] Table 5 shows that under hypoxic conditions, the relative expression levels of TGF-β1, Smad2, Smad3, Collagen II, FN, and CTGF genes in Comparative Example 1 cells increased with prolonged hypoxia. The results indicate that wild-type renal interstitial fibroblasts activate fibrosis signaling and accumulate extracellular matrix under hypoxic stimulation. The relative expression levels of TGF-β1, Smad2, Smad3, Collagen II, FN, and CTGF genes in Comparative Example 2 cells were higher than those in Comparative Example 1 cells. The results suggest that overexpression of the PDK1 gene promotes hypoxia-induced fibrosis signaling and matrix synthesis.

[0074] In Example 2, during the 12-72 hour hypoxia test period, the relative expression levels of TGF-β1, Smad2, Smad3, Collagen II, FN, and CTGF genes were lower than those in Control Example 1 cells. Combined with the glucose metabolism indicators in Example 2, knocking down the PDK1 gene blocked aerobic glycolysis reprogramming, reduced the energy and substrate required for extracellular matrix synthesis, and inhibited the TGF-β1 / Smad signaling pathway and the transcription of extracellular matrix-related proteins. The test results showed that knocking down the PDK1 gene blocked the pro-fibrotic pathway and reduced extracellular matrix synthesis and deposition under hypoxic conditions.

[0075] Test Example 6: Comparative Test of Reversal of Cell Anti-apoptotic Properties Cells from Example 2, Comparative Example 1, and Comparative Example 2 were seeded into multi-well culture plates. After cell adhesion, the multi-well culture plates were transferred to an oxygen-deficient incubator with 10% oxygen. Cells from each group were collected at 12, 24, 48, and 72 hours after hypoxia treatment. To detect the apoptosis rate, suspended cells in the culture supernatant and adherent cells detached by trypsin were collected together, centrifuged to obtain the cell pellet, and washed with pre-cooled phosphate buffer. The cells were resuspended in binding buffer, and Annexin V-APC and 7-AAD nucleic acid staining solution were added and incubated in the dark. The cell distribution ratio was detected by flow cytometry, and the total apoptosis rate was calculated. To detect gene expression levels, nucleic acid lysis buffer was added to another portion of the cell pellet to extract total RNA, which was reverse transcribed to synthesize cDNA. The relative expression levels of Caspase 3, Caspase 9, Bcl-2, and Bax genes were measured using real-time quantitative PCR, and the ratio of Bcl-2 to Bax relative expression levels was calculated.

[0076] Table 6. Apoptosis rate and relative expression levels of related apoptosis genes in different groups at different time points under hypoxia. Test Result Analysis: Hypoxic environments endow renal interstitial fibroblasts with anti-apoptotic properties. An increased Bcl-2 / Bax ratio inhibits apoptosis, while a decreased ratio promotes apoptosis; Caspase 3 and Caspase 9 proteases are involved in mediating the apoptosis execution phase.

[0077] Table 6 shows that under hypoxic conditions, the apoptosis rate of Comparative Example 1 cells decreased with prolonged hypoxia. After 72 hours of hypoxia, the apoptosis rate of Comparative Example 1 cells was 2.76%, the Bcl-2 / Bax ratio increased to 5.39, and the relative expression levels of Caspase 3 and Caspase 9 did not increase. These results indicate that wild-type renal interstitial fibroblasts exhibit an anti-apoptotic phenotype in a hypoxic environment. The apoptosis rate of Comparative Example 2 cells was lower than that of Comparative Example 1 cells, and the Bcl-2 / Bax ratio increased to 9.17. These results indicate that overexpression of the PDK1 gene enhances the anti-apoptotic ability of hypoxic cells.

[0078] In Example 2, the apoptosis rate of cells at all hypoxic time points was higher than that of cells in Comparative Example 1. After 72 hours of hypoxia, the apoptosis rate of cells in Example 2 reached 26.31%. The relative expression levels of Caspase 3 and Caspase 9 increased with prolonged hypoxia, and the Bcl-2 / Bax ratio decreased to 0.28. Knockdown of the PDK1 gene blocked aerobic glycolysis, inducing cellular energy metabolism disorders, thereby reducing the Bcl-2 / Bax ratio and activating the Caspase 9 / Caspase 3 cascade. The test results indicate that knockdown of the PDK1 gene reverses the anti-apoptotic properties of hypoxic cells and induces fibroblasts to initiate the apoptosis program.

[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A recombinant lentiviral composition for inhibiting hypoxia-induced renal fibrosis, characterized in that, The composition is made from raw materials comprising the following parts by weight: 3-5 copies of recombinant lentiviral plasmids carrying the PDK1 gene sequence; 2-4 portions of packaging aid plasmid; 1-2 copies of envelope protein expression plasmid; 0.4 to 1.0 parts of polyaluminum.

2. The recombinant lentiviral composition according to claim 1, characterized in that, The mass ratio of the recombinant lentiviral plasmid, packaging helper plasmid, and envelope protein expression plasmid is 4:3:1.

5. The PDK1 gene targeting sequence carried in the recombinant lentiviral plasmid is any one of the first, second, or third targeting sequences.

3. The recombinant lentiviral composition according to claim 1, characterized in that, The raw materials also include an additional screening component, namely puromycin, used in an amount of 0.1 to 0.2 parts.

4. The recombinant lentiviral composition according to claim 1, characterized in that, The specific components of the packaging aid plasmid and the envelope protein expression plasmid are as follows: It consists of a first packaging plasmid component carrying the gag / pol gene, a second packaging plasmid component carrying the rev gene, and a membrane expression plasmid component carrying the VSV-G gene.

5. The recombinant lentiviral composition according to claim 1, characterized in that, The recombinant lentiviral plasmid has a specific preparation method, specifically: The lentiviral backbone plasmid carrying the resistance expression cassette was linearized by double restriction endonuclease digestion and then ligated with an annealed shRNA oligonucleotide double strand targeting the PDK1 gene at 14-22°C for 1-16 hours.

6. A method for preparing a recombinant lentiviral composition, characterized in that, The preparation of a recombinant lentiviral composition as described in any one of claims 1-5 comprises the following steps: S100, plasmid co-transfection: The recombinant lentiviral plasmid, packaging helper plasmid and envelope protein expression plasmid are mixed in the specified ratio, and after adding the transfection reagent, they are co-transfected into the host packaging cells. S200, Virus Packaging and Culture: Transfected host packaging cells are continuously cultured in a medium containing fetal bovine serum, and cell culture supernatant containing virus particles is collected. S300, Concentration and Preparation: The collected cell culture supernatant is centrifuged and filtered to remove cell debris, and then polybrene in the specified ratio is added and mixed to obtain the recombinant lentivirus composition.

7. The method for preparing the recombinant lentiviral composition according to claim 6, characterized in that, In step S100, the host packaging cells are pre-treated as follows: After resuscitation, 293T cells were passaged to passages 3-5. When the cells reached the logarithmic growth phase and the cell confluence reached 70%-80%, they were washed with phosphate buffer free of calcium and magnesium ions and then used for subsequent plasmid co-transfection.

8. The method for preparing the recombinant lentiviral composition according to claim 6, characterized in that, The specific implementation method and process parameters of step S200 are as follows: The transfected cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide. The cell culture supernatant was collected twice, at 48 hours and 72 hours after transfection, and the two collections were combined.

9. The method for preparing the recombinant lentiviral composition according to claim 6, characterized in that, In step S300, the performance parameters of the obtained recombinant lentiviral composition are controlled within the range of: viral titer of 1E+8 TU / mL ~ 6E+8 TU / mL.

10. An application of PDK1 gene knockdown in inhibiting hypoxia-induced renal fibrosis, characterized in that, A recombinant lentiviral composition for inhibiting hypoxia-induced renal fibrosis as described in any one of claims 1-5, wherein the drug uses the recombinant lentiviral composition as the active ingredient to target and knock down the expression level of the PDK1 gene in renal interstitial fibroblasts. The inhibition of hypoxia-induced renal fibrosis specifically includes: Blocking the aerobic glycolytic metabolic pathway of renal interstitial fibroblasts; It inhibits the hypoxia-induced TGF-β1 / Smad profibrosis signaling pathway and reduces the synthesis and deposition of extracellular matrix-related proteins; It reverses the anti-apoptotic properties of hypoxic cells and induces apoptosis in pathologically activated renal interstitial fibroblasts.

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