EFFECT OF ULTRA-DILUTED BERBERIS VULGARIS ON NF-ΚB GENE EXPRESSION AND P38 MAPK PROTEIN SIGNALLING IN RAT RENAL TISSUE: A COMBINED QRT PCR AND WESTERN BLOT STUDY

Abstract

Background: Nuclear factor-κB and p38 mitogen- activated kinase are inflammatory and cellular stress response signalling which contributes the renal inflammation. Berberis vulgaris has the principal alkaloid called berberine, which has been found to be anti-inflammatory and antioxidant in many studies, but the effect of preparations of ultra-highly diluted form on these pathways has not been discovered. Objective: To evaluate NF-κB gene expression and p38 MAPK protein expression in rat renal tissue in an ethylene glycol-induced nephrolithiasis model, across nine experimental groups (G1–G9), following treatment with ultra-diluted Berberis vulgaris 30CH (10-60), 200CH (10-400, 1M (10-2000), 10M (10-20000), 0/1 LM Methods: This study was carried out using ethylene glycol-induced nephrolithiasis in male Wistar rats; Fifty-four male Wistar rats were selected and divided into nine groups; six rats (n=6). Groups were labelled as follows: G1(normal control), G2 as disease control (ethylene glycol only), G3, G4, G5, G6 and G7 as preventive groups with five ultra-diluted Berberis Vulgaris, G8 as a Therapeutic group and G9 as Standard drug control (Cystone). NF-κB transcripts were quantified by SYBR-Green qRT-PCR (QuantStudio 5) with GAPDH as a reference, using the 2−ΔΔCt method. RNA was extracted by the TRIzol method, DNase-treated, and reverse transcribed. Total p38 MAPK protein was assessed using SDS-PAGE/western blot, and band intensity was quantified using ImageJ after normalising to β-actin. Data are expressed as mean ± SD and compared by one-way ANOVA; p < 0.05 was considered significant. Before statistical analysis, normality for each experimental group was tested using the Shapiro–Wilk test. All groups showed a normal distribution (p > 0.05), indicating that the assumption of normality was met. Homogeneity of variances was subsequently evaluated using Levene's test, confirming equal variances among groups (p > 0.05). Therefore, one-way analysis of variance (ANOVA) was performed, followed by Tukey's multiple comparisons test for pairwise group comparisons. Statistical analyses were conducted using SPSS SOFTWARE (Version 2026), and a two-tailed p < 0.05 was considered statistically significant. Results: One-way ANOVA showed a highly significant difference in p38 MAPK protein expression among the nine groups (F (8,45) = 264.77, p < 0.0001) and in NF-κB gene expression (F (8,45) = 3.73, p = 0.00203). The disease control (G2) showed the highest NF-κB (RQ = 2.323) and p38 MAPK (33.8 ± 1.4%) expression, consistent with activation of inflammatory signalling by ethylene glycol. Treatment groups showed reduced expression of both markers relative to disease control, with the greatest suppression in G6 (10M: NF-κB RQ = 0.097; p38 MAPK = 18.1 ± 0.7%) and G3 (30CH: RQ = 0.026; p38 MAPK = 17.2 ± 2.1%). The values of both the inflammatory markers NF-κB and p38 MAPK are strongly expressed and correlated among the groups (Pearson r = 0.88, p = 0.0016). Conclusion: Ultra-diluted Berberis vulgaris shows modulatory changes in the inflammatory pathway, NF κB transcription and p38 MAPK protein expression in renal tissue. Because ultra-dilutions beyond the Avogadro limit contain no molecules of the source substance, this association should be interpreted as control-referenced and hypothesis-generating; independent replication with a succussed-vehicle comparator is warranted before mechanistic or therapeutic claims are drawn.