| ObjectiveTo make a preliminary study on the accumulation of heavy metal Hg in blood and kidney after the long-term use of the Tibetan medicine Tsothel as well as the potential nephrotoxicity of Tsothel in the hope of revealing the possible nephrotoxicity and action mechanism of the Tsothel to provide some theoretical basis for the clinically safe, reasonable and efficient use of Tsothel.Methods(1) The acute toxicity test on KM mice and SD rats was conducted in reliance on maximum dosage with a view to carrying out the preliminary exploration on the safety of the Tibetan medicine Tsothel;(2) Inductively coupled plasma mass spectrometry (ICP-MS) was used to test the Hg contents in the blood and kidney of the rats 90 days,135 days, and 180 days after medication as well as 30 days and 120 days after drug withdrawal in order to investigate into the dynamic variation rules of Hg in animal blood and its accumulation in kidney.(3)En2yme-linked immunosorbent assay (ELISA) was applied to testing the content or activity of the SCr and BUN in the blood of the rats, and of the Kim-1, NAG, RBP, and P2-MG in the urine, and of the Kim-1, MT, GSH, GSH-Px, MDA, SOD in the kidney of the rats 90 days,135 days, and 180 days after medication as well as 30 days after drug withdrawal. Additionally, the GSH content in rat’s kidney tissue was re-tested 120 days after drug withdrawal with a view to probing into the influence that the Hg accumulated in animal after long-term use of the Tibetan medicine Tsothel exercised on the protein or enzyme level related to renal injury in animal blood, urine and kidney;(4) The real-time quantitative fluorescence was utilized to investigate into the influence of Tsothel on the relative expression levels of the Kim-1, MT-1, MT-2, GST-pi in rat’s kidney, as well as the relative expression level of GST-pi mRNA 120 days in rat’s kidney after drug withdrawal;(5) The methods such as HE staining, immunohistochemistry, and flow cytometry were applied to testing the kidney coefficient, renal pathology, renal Ki 61 protein expression, renal cell cycle, and apoptosis rate of rats 90 days,135 days and 180 days after medication as well as 30 days after drug withdrawal with the purpose of investigating into the influence of the Tibetan medicine Tsothel over the cell morphology, cell proliferation activity, cell cycle and apoptosis rate of renal tissue.Results(1) The KM mice and SD rats were dosed with the Tibetan medicine Tsothel at a time within one day based on the maximum concentration and volume (4498 times of 0.667mg/kg-the daily clinical recommended dose for the adults); after the observation for 14 consecutive days, Tsothel was preliminarily found without falling into the category of acute toxic agent according to the normal appearance, physical sign and activities, and the gradually increased weight of the rats.(2) After the continuous medication for 180 consecutive days, the blood Hg and renal Hg saw an increase in content with the passage of administration time, and heavily rely on dosage.; compared with renal Hg, blood Hg was metabolized faster and able to return to normal upon drug withdrawal, whereas despite the remarkable decline in content, renal Hg was still accumulated in large amount; therefore, according to preliminary verification, the major target organ of Hg in Tsothel was kidney.(3) 180 days after continuous medication and 30 days after drug withdrawal, the Tsothel exercised an insignificant influence on Kim-1, NAG, RBP, and β2-MG in the urine of rats, Scr and BUN in the serum, renal tissue MDA and SOD. However,90 days and 180 days after medication, Kim-1, MT, GSH-Px and GSH in kidney faced some changes, and 30 days after drug withdrawal, other indices returned to normal reversibly except that GSH activity was on the high side. GSH’s activity also returned to normal 120 days after drug withdrawal.(4) 180 days after continuous medication with the Tibetan medicine Tsothel, the relative expression levels of the MT-1 mRNAin all drug-given groups have improved significantly; for the low-and medium-dose groups, the relative expression levels of Kim-1 and GST-Pi mRNA have improved significantly, but the relative expression level of MT-2 mRNAsuffered a decline.30 days after drug withdrawal, the relative expression levels of Kim-1, MT-1, and MT-2 mRNA have returned to normal and become insignificantly different from those of the blank control group, whereas the relative expression level of the GST-Pi mRNA was still on the high side, which is predicted to be the stress detoxification manifestation of detoxification system in the face of exogenous material stimulation.120 days after drug withdrawal, the expression level of GST-Pi mRNA basically returned to normal. The value of the low-dose group during drug delivery rose obviously, which is possibly associated with organism’s multi-way regulatory mechanism. Its specific mechanism needs further study.(5) 180 days after consecutive medication and 30 days after drug withdrawal, the renal coefficient, renal histopathology, renal Ki67 protein expression, renal cell cycle and apoptosis rate of the rats in all groups faced insignificant changes, which preliminarily proves that the Tibetan medicine Tsothel has insignificant influence over the cell morphology, cell proliferation activity, cell cycle and apoptosis rate of rats’ renal tissue. Conclusion(1) Tibetan medicine Tsothel has no significant acute toxicity;(2) Long-term use of the Tibetan medicine Tsothel may lead to the rise in blood Hg of rats, the accumulation of much Hg in kidney, and the residuals after long-term drug withdrawal. It is predicted that Tsothel is permanently and irrevocably accumulated in kidney;(3) The long-term use of substantial Tsothel will be likely to cause the potentially reversible injury to kidney. This mechanism of action is probably closely associated with the stress detoxification reaction made by the detoxification system activated by the influence on content or activity of Kim-1, MT, GSH, and GSH-Px, and relative expression levels of Kim-1, MT, and GST-Pi mRNA;(4) The clinical use of Tsothel requires that not only its use time and dosage should be controlled, but relevant indices of blood Hg and renal function should be tracked and monitored. Meanwhile, there is also a need to conduct an in-depth study on whether Hg residuals in kidney will further damage organisms. |
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